JPH078236B2 - Animal cell culture method and centrifuge - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for culturing animal cells and a centrifuge for separating animal cells. More specifically, the present invention relates to a method for culturing animal cells and a separation device for culturing animal cells in a suspended state.

(B) Prior art In suspension culture of cells, a new culture solution is generally supplied to the culture tank in order to prevent the growth and proliferation of fine particles from stopping at a relatively low density and to culture the cells in a large amount and at a high density. Meanwhile, a method of culturing while discharging an old culture solution containing a growth-inhibiting substance to the outside of the culture tank, that is, a method commonly called a perfusion method has been proposed (Annu
al Reports on Fermentation Processes Vol.6).

In culturing using this method, it is important to efficiently separate the living cells in the suspension liquid from the old culture liquid, remove the old culture liquid from the culture tank, and grow the cells in the culture tank. Maintaining the environment under optimal conditions. Various filters and various types have been proposed to separate the live culture and the old culture from the suspension culture, but as an industrial device in terms of filter clogging and the complexity of the structure of the device. It is hard to say that all of them are satisfactory.

Japanese Patent Application Laid-Open No. 63-252558 proposes an animal cell separating apparatus and a 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. There is concern about damage to cells due to the pressure difference in the centrifuge that occurs when cells pass through the large liquid, and it cannot be said to be suitable for all cells.

Further, since only a part of the main body of the centrifugal separator can be used, there is a drawback that the volumetric efficiency is low.

(C) Object of the Invention The object of the present invention is to separate animal cells from a suspension culture solution in a centrifuge alive, and then separate and accumulate the animal cells from a centrifuge in a specific liquid medium. It is an object of the present invention to provide an industrially advantageous culturing method in which separation and extraction are alternately carried out.

Still another object of the present invention is to provide a centrifuge suitable for use in the above method of the present invention.

(D) Structure of the Invention That is, the present invention comprises: (A) subjecting an animal cell to suspension culture in a culture tank, (B) extracting a portion of a suspension culture solution containing animal cells from the culture tank, and (C) ) The extracted suspension culture solution is continuously supplied to the centrifugal separation space of the rotating centrifugal separator through a supply port provided at a position where the centrifugal force is greater, for a certain period of time, and the centrifugal force is further increased. The mother liquor separated from the animal cells is continuously taken out for a certain period of time from an outlet provided at a position where it acts small, and the animal cells are accumulated in the above-mentioned centrifugal separation void, and (D) water is immiscible and A liquid medium that has a higher density than both cells and culture medium and that does not inhibit the growth of animal cells is supplied from the supply port while the centrifuge is rotating, and accumulated in the centrifugation space from the discharge port. It Culturing animal cells, wherein said animal cells are extruded with said liquid medium together with mother liquor and withdrawn, and (E) at least a part of said taken out animal cells is returned to said culture tank for said step (A). Is the way.

The method for culturing animal cells of the present invention 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 not only natural animal cells but also cells that have been artificially or genetically modified, such as hybridomas. Lymphocyte-producing cells that produce lymphokines such as IL-2, interferon (IF
N) diploid cells that produce useful bioactive substances such as
Alternatively, it can be 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 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. 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.

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

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.

As a matter of course, by maintaining the concentration of the substance that inhibits the growth of animal cells at a low level under sufficient control as described above, culturing is performed while maintaining the increased growth density of animal cells for a long period of time. It can be continued, and an increased production of useful metabolites can be secured.

The above culturing method comprises the step (c) of accumulating live animal cells in the centrifuge cavity of the centrifuge, and then, in the step (D), the accumulated live animal cells are not mixed with water. It is characterized in that it is extruded and extracted together with the mother liquor in a liquid medium having a density higher than that of both animal cells and culture medium and not inhibiting 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 for a certain period of time into the centrifugation space of the centrifuge,
Moreover, it is necessary to continuously remove the mother liquor separated from the living animal cells for a certain period of time. That is, the suspension culture medium extracted in the step (B) is supplied to the centrifugal separation space until a desired amount of animal cells are accumulated because the concentration of animal cells is low. The operating conditions of the centrifuge at that time are: (1) θ ≦ 300 (2) × θ ≦ 3 × 10 ⁴ (3) Q / S · ≦ 0.3 and (4) 5 ≦≦ 2000 It is desirable to adopt.

Average residence time of animal cells in the centrifuge (θ,
Minutes) should be kept below 300 minutes, as shown in equation (1) above. When it exceeds 300 minutes, the viability of animal cells is significantly reduced due to the cause of oxygen deficiency in the centrifuge. The average residence time (θ) is preferably 150 minutes or less, in particular 60 minutes or less.

Average retention time of animal cells in the centrifuge (θ)
For example, in a continuous method, that is, in a system in which a suspension culture solution is continuously charged into a centrifuge and separated animal cells are continuously taken out, the animal cells in the prepared suspension culture solution may exist under centrifugal conditions. It is determined as a value obtained by dividing the volume of the space (V · cm ³ ) by the removal rate (Qc · cm ³ / min) of the separated animal cell-containing component from the centrifuge.

Is the centrifugal effect when the centrifugal separation operation is performed. The distance from the rotation axis is γ (cm) and the rotation angular velocity is ω (radian / sec).
If the acceleration of gravity is expressed in g (cm / sec ² ), γω ²
Expressed as / g. The centrifugal effect indicates, so to speak, the magnitude of the centrifugal force applied to animal cells, and therefore the position of the culture solution supply port for supplying the suspension culture solution to be separated to the centrifuge (distance γ from the rotation axis ).

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 () is preferably in the range from 10 to 1000, particularly preferably in the range from 20 to 300.

In addition, the value (.θ) obtained by multiplying by 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 θ is particularly preferably 2 × 10 ⁴ or less.

Further, S (cm ² ) is the sedimentation area (cm ² ) when the centrifugal force acts. S (cm ² ) is the position of the supply port of the suspension culture solution to be separated to the centrifuge (distance γ from the rotation axis
(Cm)) is defined as the effective area involved in the separation. When the virtual circle at the position of the supply port at a distance γ from the rotation axis does not intersect with the sinking surface, the effective area is
The value of IIγ ² h is obtained by the position (γ) of the supply port and the height (h) of the liquid surface of the suspension culture solution at that position. 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 IIγ ² h is multiplied by the angle ratio until the intersection. Is required as. 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.

The value S · Z obtained by multiplying the effective area S and the centrifugal effect Z is
It is a parameter indicating the separation ability of the centrifugal separator. In the method of the present invention, it is necessary to set the value obtained by dividing the amount Q (ml / min) of the suspension culture solution supplied to the centrifugal separator per unit time by this parameter, that is, the value Q / S · Z to 0.3 or less. . The value of Q / S · Z is preferably 0.2 or less, particularly preferably 0.1 or less.

Thus, by ensuring the operating conditions of (1) to (4) above, according to the step (C) of the present invention, the animal cells can be efficiently separated from the suspension culture solution with a very high survival rate in a living state. Is possible.

As a centrifuge, as described above, it is convenient to continuously supply the suspension culture solution and continuously withdraw the mother liquor for a certain period of time, so that the centrifuge cavity has a supply port and a discharge port, and the supply port is a centrifuge. A force is applied to a position where a larger force is applied, and a discharge port is provided to a position where a centrifugal force is smaller.

As such a centrifuge device, according to the invention, the centrifuge cavity has an outer peripheral wall which is inclined in a direction towards the central axis of the rotor at an angle of 50-90 ° to the direction perpendicular to the central axis of the rotor. A peripheral slot, the outer peripheral wall of the peripheral slot defining a helix with respect to the center axis of the rotor in an imaginary plane transverse to the center axis of the rotor, and the peripheral slot being located furthest from the center axis of the rotor. A centrifuge characterized by having a supply port in the vicinity thereof or in the vicinity of the central axis of the rotor or in the vicinity thereof is preferably provided.

In the centrifuge, the centrifuge cavity for receiving the suspension culture consists of a peripheral slot provided in the rotor. The peripheral slots are 50 to 50 ° to the direction perpendicular to the center axis of the rotor in the direction toward the center axis of the rotor.
It has a sloping outer peripheral wall at an angle of 90 °, preferably at an angle of 60-85 °. By having such an outer peripheral wall inclined in the central axis direction of the rotor, it becomes possible for living animal cells separated from the mother liquor to move smoothly on the outer peripheral wall in a direction away from the central axis of the rotor. The outer peripheral wall sloping at the angle may occupy all or part of the outer peripheral wall of the peripheral slot. When occupying the whole, the angle of the outer peripheral wall may be uniform throughout, or may be a combination of two or more angles within the angle values in the above range. When it occupies a part, it preferably comprises a wall having an angle value in the above range and a wall inclined at an angle smaller than 50 ° with respect to the direction perpendicular to the central axis of the rotor.

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 edge wall is preferably in the direction toward the central axis of the rotor, with respect to the direction orthogonal to the central axis of the rotor, like the outer peripheral wall, at 50 to 90 °, more preferably 60 °.
It is inclined at ~ 85 °. In addition, the inner peripheral wall can define an arc or spiral, preferably a spiral, with respect to the center axis of the rotor in an imaginary plane that intersects the center axis of the rotor.

In addition, the peripheral slots have 360 degrees around the center axis of the rotor.
It can exist within ° and can exist over 360 °. If it exists over 360 °,
The peripheral slots can be spiral-wound around the center axis of the rotor in the same virtual plane, i.e. without changing the angle in the direction of the center axis of the rotor, and also around the center axis of the rotor. It can exist in a spiral shape inclined at an angle in the direction of the central axis. The length of the peripheral slot is 36 mm around the center axis of the rotor, as described above.
Providing a peripheral slot beyond 0 ° allows for a longer length, which allows a slow and smooth in-vivo separation of animal cells. 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.

The centrifuge device has a supply port for supplying the suspension culture solution to the centrifuge void, that is, the peripheral slot, during operation of the centrifuge device, that is, during rotation of the rotor, in order to extract the mother liquor separated from the animal cells. And a discharge port. 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, and the discharge port is provided at a position closest to or near the central axis of the rotor of the peripheral slot.

The viable animal cells separated by the centrifuge as described above and accumulated in the centrifuge void are, according to the method of the present invention, then, in step (D), added to the operating centrifuge, From the supply port, instead of the suspension culture solution, a liquid medium that is immiscible with water and has a higher density than both animal cells and culture solution and that does not inhibit the growth of animal cells is supplied from the discharge port. And is extruded with the liquid medium.

Perfluorocarbon, for example, is preferably used as the liquid medium.

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 perfluoroalkanes having 8 or more carbon atoms,
Perfluorocycloalkanes (eg, perfluorodecalin, perfluoromethyldecalin, carbon number 3-5)
Perfluoroalkylcyclohexane having an alkyl substituent), perfluoroalkyltetrahydrofuran having an alkyl substituent having 5 to 7 carbon atoms, and 4 carbon atoms
Examples thereof include perfluoroalkyltetrahydropyrans having 6 alkyl substituents and perfluoroadamantanes (eg, perfluoroadamantane, perfluoromethyladamantane, perfluorodimethyladamantane, perfluoromethylethyladamantane, perfluorodiethyladamantane, etc.). 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 centrifugal separation space is constantly washed with the liquid medium, and therefore the inside of the centrifugal separation space is washed. Is always maintained in an environment suitable for living animal cells, and it is possible to create a situation suitable for continuous operation that is industrially advantageous.

According to the culturing method of the present invention, at least a part of the proliferating viable animal cells removed in step (D) is then added in step (E) to the culture tank for step (A). Returned to.

The above culturing method of the present invention provides an industrially advantageous method that exhibits the above-mentioned advantages by repeating steps (B) to (E).

Hereinafter, the present invention will be further described with reference to specific embodiments by the above-mentioned culture method and centrifuge.

FIG. 1 discloses a series of devices suitable for carrying out the culture method of the present invention.

In the apparatus shown in FIG. 1, the culture tank AP-1 is equipped with a new culture medium inlet A, an air (O ₂ ) introduction pipe B, an oxygen concentration in the air introduced by measuring the oxygen concentration in the culture solution in the culture tank. For adjusting
It is equipped with a DOC, an agitator ST, an exhaust pipe C, and a suspension culture liquid extraction conduit D. Extraction conduit D is pump P
-I is connected to the centrifuge AP-2.

At the bottom of the culture tank AP-1, there is a conduit H for withdrawing the phase LQ of the liquid substance, and this conduit H goes to the centrifuge AP-2 at point P via pump P-II. It is connected to an onward conduit.

A pipe J leading to the culture tank AP-1 is connected to the point P through a valve Z. At the point Q, the conduits E, F, G are connected to the conduit extending from the centrifuge AP-2.

The conduit E is connected to the culture tank AP-1 through the valve X, the conduit F is connected to the storage tank AP-3 through the valve Y, and the conduit G is connected to the pump P-II.
Each is connected to a new medium tank AP-4 through I.

A new culture medium is charged into the culture tank AP-1, air is introduced from the air introduction tube B, the stirrer is rotated, and the animal cells are seeded to start the culture. After culturing for a predetermined time, when the number of cells in the culture tank increased to a saturated state, rotation of the pump P-I was started, and the culture solution in the culture tank was sent to the centrifuge AP-2 through the conduit D. The mother liquor is separated from the animal cells by a centrifuge, and the separated mother liquor is continuously sent to the storage tank AP-3 through the conduit F in which the valve Y is opened.

During this period, new medium is introduced into the culture tank continuously or intermittently from the medium charging port A so that the liquid level in the culture tank AP-1 does not change significantly. After operating the centrifuge AP-2 for a certain period as described above, the operation of the pump P-I is stopped, the valve Y is closed and the valve X is opened,
Then, the operation of the pump P-II is started. Thereby,
The liquid substance at the bottom of the culture tank is introduced into the centrifuge AP-2 through the conduit H, and the live animal cells accumulated in AP-2 are extruded and returned to the culture tank through the conduit E together with the cells. By performing this operation while the centrifuge is operated, the liquid substance is retained in the centrifuge, and the cells retained until then are naturally discharged due to the difference in specific gravity. At this time, part or all of the liquid substance may be introduced into the culture tank AP-1, so that the phase LQ of the liquid substance 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 LQ formed at the bottom of the fermentor is of great advantage for cultivation on an industrial scale.

After operating for a certain period as described above, the operation of the pump P-II is stopped, the valve X is closed, the valve Z is opened, and then the operation of the pump P-III is started. Thereby, the liquid substance in the centrifuge returns to the culture tank. By such an operation, the cells do not pass through the liquid substance 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 the liquid substance can be eliminated or extremely reduced. If the pressure applied to the cells is substantially 0.2 kgf / cm ² or less, there is little damage, so it is acceptable to pass through a liquid substance corresponding to that.

Further, by continuing the operation of the 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.

FIG. 2 shows a conceptual diagram of another apparatus suitable for carrying out the above-mentioned culture method of the present invention.

In FIG. 2, 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 provided on the high centrifugal force side of the centrifugal separator S,
Animal cells are separated from the suspension culture solution by centrifugal force, and the culture mother liquor from which the animal cells have been separated is continuously and aseptically tanked from the mother liquor discharge port provided on the low centrifugal force side.
Take it out to T2. Then, from the bottom of the culture tank T1, a liquid having no affinity for cells and culture liquid and having a high density is extracted by a pump P2 from a supply port provided on the high centrifugal force side of the centrifugal separator S. By supplying a fixed amount, the culture solution containing the separated cells in the peripheral slot is aseptically taken out from the animal cell take-out port provided on the low centrifugal force side of the centrifuge and returned to the culture tank T1. After that, the cell-free medium is extracted from the tank T3 by the pump P3 and supplied from the cell outlet of the centrifuge S to return the liquid having a high density in the centrifuge to the culture tank T1 again, and to close the peripheral slot. Replace with medium. The tank T4 is a new medium containing no cells, and is continuously or intermittently supplied to the T1 by the pump P4 so that the liquid surface level of 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. It will not be necessary to explain that the valves V1 to V6 need to be opened and closed to suit the above operation.

FIG. 3 shows a schematic partial perspective view of a suitable centrifuge for use in the above method, in which a peripheral slot 23 is formed by both the inner peripheral wall 21 and the outer peripheral wall 22. There is. 24 is a suspension culture solution supply port and a high-density liquid outlet, 25 is a cell-free culture mother liquor and a culture medium containing separated cells, and 26 and 27 are rotor bodies. A nozzle for supplying to and / or removing from the rotor body.

Further, the angle α between the slot surface and the centrifugal direction shown in FIG.
90 ° range is good. The depth d of the peripheral slot is preferably in the range of 3-5 mm. Also, the inner wall surface of the plate-shaped peripheral slot may be subjected to a hydrophobic treatment in order to prevent the attachment of cells.

As the hydrophobic treatment, for example, a formula in which a wall surface is coated with a fluororesin such as PTFE (tetrafluororesin) is suitable.

The angle α ′ between the upper side of the peripheral slot 23 and the centrifugal direction shown in FIG.
Is preferably in the range of 5 to 10 °. The angle α ″ between the lower side of the slot and the centrifugal direction is preferably in the range of 0 to 5 °.

FIG. 4 shows a schematic partial perspective view of another embodiment of the centrifugal separator of the present invention.

In FIG. 4, the peripheral slot 31 is formed by being closed by a plate 28 and flat plates 29, 30 which are inclined and are wound in a spiral shape at equal intervals. Reference numeral 32 serves both as a suspension culture solution supply port and a high-density liquid extraction port, and is connected to the rotating shaft 34. 33 is a cell-free culture mother liquor and the separated cells are an outlet of the culture fluid,
It is connected to the rotary shaft 35. 36, 37 are nozzles for supplying to and / or taking out from the rotor body. In addition, the angle α between the slot surface and the centrifugal direction shown in FIG.
Is preferably in the range of 50 to 90 °. The width d of the spiral peripheral slot is preferably in the range of 3-5 mm.

Further, the inner wall surface of the slot may be subjected to a hydrophobic treatment in order to prevent the attachment of cells. A separator plate may be provided in the peripheral slot to further improve the separation efficiency.

FIG. 5 shows a schematic partial perspective view of still another embodiment of the centrifugal separator of the present invention.

In FIG. 5, a peripheral slot 40 is formed by both the inner peripheral wall 38 and the outer peripheral wall 39. 41 is a suspension culture solution supply port and a high-density liquid outlet, 42 is a cell-free culture mother liquor and a culture medium containing separated cells, and 43 and 44 are rotor bodies A nozzle for supplying to and / or removing from the rotor body.

Further, the angle α between the slot surface and the centrifugal direction shown in FIG.
90 ° range is good. The depth d of the peripheral slot is preferably in the range of 3-5 mm.

Further, it is not necessary to further explain that the hydrophobic treatment on the inner wall surface of the plate-shaped peripheral slot is effective.

(E) Effect of the Invention According to the present invention, it is possible to efficiently separate live cells in a suspension liquid and old culture liquid without damaging the cells, and it is possible to industrially excellent cell culture. It was

(F) Examples Hereinafter, the present invention will be described in detail with reference to Examples.

Example 1 (1) Culture device The culture system shown in the attached FIG. 1 was used. Culture tank (AP-
1) is a glass-made agitation type culture tank having a total volume of 2, and the net amount of the culture solution charged is 1.2.

Fluorocarbon at the bottom of the culture tank
FLUORINERT FC-40 ) Was put in 300 ml.

AP-2 in FIG. 1 is a centrifuge having a rotor having a sedimentation area S = 31.4 cm ² , an effective volume of 11 ml, and a turning radius of 10 cm. Pumps P-I, P-II, P-III are peristaltic pumps.

(2) Culture medium As the 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 1 in the above basal medium
0 μg / ml, ethanolamine 10 μg / ml, selenite 2 × 10 ^-6 m
ole / supplemented was used as the medium.

(3) Culture method and result The culture system was autoclaved in advance. Then, medium 1.2 that had been over-sterilized was charged into a culture tank.
Then, 5 × 10 5 mouse × human hybridoma × 87 strains obtained by fusing the mouse myeloma P3U1 strain with human B cells were obtained.
^The cells were seeded at ⁵ cells / ml. This cell produces IgG. In the culture tank, oxygen gas containing 5% of carbon dioxide gas is automatically controlled and introduced through the blowing nozzle B so that the dissolved oxygen concentration in the culture solution becomes 3 ppm. A marine-type stirring blade is attached to the culture tank, and the stirring speed is 60 rpm.

Batch culture was performed for 3 days after seeding.

As shown in Table 1, the cell density reached 1.0 × 10 ⁶ cells / ml on the 3rd day after the start of the culture (after 24 hours), and it was judged that the maximum density was reached in the batch culture, and the perfusion culture using a centrifuge was performed. Started. That is, the centrifuge in which the over-sterilized culture solution was placed was driven and the rotation speed was adjusted so that the centrifugal effect was 100 g.

Next, with valves X and Z closed, pumps P-II and P-III stopped, and valve Y open, pump P-I was driven to centrifuge 200 ml of the culture mixture at a speed of 20 ml / mm for 10 minutes. It was sent to. The culture medium separated from the cells by the centrifuge is line F
Through the storage tank AP-3 for storing the old culture solution. The cell density in this was 5.0 × 10 ⁴ cells / ml.

After the above operation is completed, the valve X is opened, the valves Y and Z are closed, the pumps P-I and P-III are stopped, and the pump P-
By driving II, 50 ml of fluorocarbon at the bottom of the culture tank AP-1 was fed at a rate of 5 ml / min. By this operation, the cells retained in the centrifugal rotor were discharged together with the fluorocarbon from the centrifugal separator, and returned to the culture tank AP-1 via the line E. At this time, the maximum pressure applied to the cells in the centrifugal rotor was 0.05 kgf / cm ² .

Next, valve Z is opened, valves X and Y are closed, and pumps P-I and P-
With II stopped, the pump P-III was driven, the new medium was sent to the centrifuge AP-2, and the fluorocarbon in AP-2 was returned to the culture tank AP-1.

This operation was automatically performed once every time shown in Table 1, and the culture was continued. The new medium was continuously supplied to the culture tank from the line A so that the liquid level in the culture tank was constant on average.

In the above experiment, Q / (S ・ Z) is 0.032 cm / min and θ is 12.
It was 5 minZ · θ = 250.

Table 1 shows a part of the experimental conditions and the experimental results.

Comparative Example 1 When the flow path in FIG. 1 was changed and cells were taken out from the centrifugal rotor, fluorocarbon and new medium were put into the separation layer through an opening close to the rotation axis of the rotor, and the separated cells were opened far from the rotation axis of the rotor. I took it out from the department.

All other conditions were the same conditions × 87 strains were cultured.

In this culture method, the pressure applied to the cells is 0.1 kgf.
/ cm ² and the maximum cell density reached is 30% higher than that in Example 1.
Diminished. The antibody concentration also decreased by 30%.

Observation of the cells taken out from the centrifugal rotor with a microscope revealed that the cells were damaged as compared with the case of the example.

Example 2 (1) Culture device A culture system having a cell separation system shown in the attached FIG. 2 was used. The culture tank (T1) is a glass-made agitation culture tank having a total volume of 2, and the net amount of the culture solution charged is 1.2.

Fluorocarbon at the bottom of the culture tank
FLUORINERT FC-40 ) Was put in 300 ml.

The centrifuge used was a centrifuge having a rotor having a sedimentation area S = 130 cm ² , an effective volume of 75 ml and a turning radius of 10 cm shown in FIGS. 3 and 4. Pumps P1, P2, 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 1 in the above basal medium
0 μg / ml, ethanolamine 10 μg / ml, selenious acid 2 × 10
^-6 mole / added was used as the medium.

(3) Culture method and result The culture system was autoclaved in advance. Then, medium 1.2 that had been over-sterilized was charged into the culture tank T1. Then, a mouse x human hybridoma x 87 strain obtained by fusing the mouse myeloma P3U1 strain and human B cell was used as 3.
The cells were seeded at 3 × 10 ⁵ cells / ml. This cell is IgG
Is to produce. In the culture tank, oxygen gas containing 5% of carbon dioxide gas is automatically controlled and fed through the 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 attached to the culture tank, and the stirring speed is 60 rpm.

Batch culture was performed for 3 days after seeding.

As shown in Table 2, the cell density reached 5.8 × 10 ⁵ cells / ml on the 3rd day after the start of the culture (after 24 hours), and it was judged that the maximum density was reached in the batch culture, and the perfusion culture using a centrifuge was performed. Started. That is, the centrifuge in which the over-sterilized culture solution was placed was driven, and the rotation speed was adjusted so that the centrifugal effect was 80 g.

Then, 200 ml of the culture mixture was transferred to the centrifuge S at a speed of 20 ml / mm for 10 minutes. The culture mother liquor separated from the cells by the centrifuge flowed out and was stored in the old culture medium storage tank T2. The cell density in this was 4.0 × 10 ⁴ cells / ml.

After the above operation is completed, drive the pump P2 and
120 ml of the fluorocarbon at the bottom of the column was sent to the centrifuge S at a rate of 30 ml / min for 4 minutes. By this operation, the cells retained in the centrifugal rotor were discharged together with the fluorocarbon from the centrifugal separator, and returned to the culture tank T1. At this time, the maximum pressure applied to the cells in the centrifugal rotor is
It was 0.05 kgf / cm ² .

Next, the pumps P1 and P2 were stopped, the pump P3 was driven, the new medium in T3 was sent to the centrifugal separator S, and the fluorocarbon in the centrifugal separator was returned to the culture tank T1.

This operation was automatically performed once every time shown in Table 2 to continue the culture. So that the liquid level in the culture tank is constant on average
Fresh medium in T4 was continuously fed to the fermentor.

Table 2 shows a part of the experimental conditions and the experimental results.

Example 3 (1) Culture device A culture system having a cell separation system shown in the attached FIG. 2 was used. However, the culture tank (T1) is an agitated culture tank made of glass and having a total volume of 15, and the net amount of the culture solution charged is 10.

Fluorocarbon at the bottom of the culture tank
FLUORINERT FC-40 ) Was put in 900 ml.

The centrifuge has a sedimentation area S = 130 cm ² in Fig. 4 and an effective working volume of 7
A centrifuge with a rotor of 5 ml and a turning radius of 10 cm was used. Pumps P1, P2, P3 are peristaltic pumps.

(2) Medium As a basal medium, a mixture of RPMI1640 medium, Ham-12 medium and Dulbecco's modified Eagle medium in a ratio of 2: 1: 1 (hereinafter referred to as RDF) was used.

Insulin 9 μg / ml, transferrin 1 in the above basal medium
0 μg / ml, ethanolamine 10 μg / ml, selenious acid 2 × 10
^-6 mole / added was used as the medium.

(3) Culture method and result The culture system was autoclaved in advance. Thereafter, the medium 10 that had been over-sterilized was charged into the culture tank T1.
Then, a mouse x human hybridoma x 87 strain obtained by fusing the mouse myeloma P3U1 strain and human B cell was converted to 6.2 x 1
The cells were seeded at 0 ⁵ cells / ml. This cell produces IgG. In the culture tank, oxygen gas containing 5% of carbon dioxide gas is automatically controlled and fed through the 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 attached to the culture tank, and the stirring speed is 30 rpm.

Batch culture was performed for 3 days after seeding.

As shown in Table 3, the cell density reached 9.8 × 10 ⁵ cells / ml on the first day after the start of the culture (after 24 hours), and it was judged that the maximum density was reached in the batch culture, and the perfusion culture using a centrifuge was performed. Started. That is, the centrifuge in which the over-sterilized culture solution was placed was driven, and the rotation speed was adjusted so that the centrifugal effect was 80 g.

Then, 500 ml of the culture mixture was transferred to the centrifuge S at a speed of 50 ml / min for 10 minutes. The culture mother liquor separated from the cells by the centrifuge flowed out and was stored in the old culture medium storage tank T2. The cell density in this was 1.0 × 10 ⁴ cells / ml or less.

After the above operation is completed, drive the pump P2 and
120 ml of the fluorocarbon at the bottom of the column was sent to the centrifuge S at a rate of 30 ml / min for 4 minutes. By this operation, the cells retained in the centrifugal rotor were discharged from the centrifugal separator together with the fluorocarbon, and returned to the culture tank T1. At this time, the maximum pressure applied to the cells in the centrifugal rotor was 0.05 kgf / cm ² .

Next, the pumps P1 and P2 were stopped, the pump P3 was driven, the new medium in T3 was sent to the centrifuge S, and the fluorocarbon in the centrifuge was returned to the culture tank T1.

This operation was automatically performed once every time shown in Table 3 to continue the culture. So that the liquid level in the culture tank is constant on average
Fresh medium in T4 was continuously fed to the fermentor.

Table 3 shows a part of the experimental conditions and the experimental results.

[Brief description of drawings]

1 and 2 are schematic diagrams of a series of devices suitable for carrying out the culture method of the present invention. FIG. 3 is a schematic partially transparent perspective view of an embodiment of the centrifugal separator of the present invention. 4 and 5 are schematic partial perspective views of still another centrifugal separator of the present invention.

Claims (11)

[Claims] 1. (A) An animal cell is subjected to suspension culture in a culture tank, (B) a part of the suspension culture solution containing the animal cell is withdrawn from the culture tank, and (C) the extracted suspension culture solution. Is continuously supplied to the centrifugal separation space of the rotating centrifugal separator from a supply port provided at a position where centrifugal force is greater, and is provided at a position where centrifugal force is smaller. The mother liquor separated from the animal cells is continuously taken out from the discharge port for a certain period of time to accumulate the animal cells in the above-mentioned centrifugation space, and (D) is immiscible with water, A liquid medium having a high density and not inhibiting the growth of animal cells is supplied from the supply port while rotating the centrifuge, and the animal cells accumulated in the centrifugation space from the discharge port are used as mother liquor. Tomo A method for culturing animal cells, characterized in that (E) at least a part of the taken out animal cells is returned to the culture tank for the above step (A) by extruding with the liquid medium and extracting. 2. The culture method according to claim 1, wherein the steps (B) to (E) are repeated. 3. The centrifugal separator is operated under the following operating conditions: (1) θ ≦ 300 (2) × θ ≦ 3 × 10 ⁴ (3) Q / S · ≦ 0.3 and (4) 5 ≦≦ 2000 The method of claim 1, wherein the method is operated at. 4. The centrifuge void comprises a peripheral slot having an outer peripheral wall that is inclined in a direction toward the central axis of the rotor and at an angle of 50 to 90 ° with respect to a direction perpendicular to the central axis of the rotor, the peripheral slot. The outer peripheral wall of the slot defines a spiral with respect to the center axis of the rotor in an imaginary plane transverse to the center axis of the rotor, and the peripheral slot defines a feed port at or near a position furthest from the center axis of the rotor. A centrifuge, characterized in that it has an outlet at a position closest to the central axis of the rotor or in the vicinity thereof. 5. The method wherein the outer peripheral wall of the peripheral slot is perpendicular to the center axis of the rotor in a direction toward the center axis of the rotor.
The centrifuge according to claim 4, comprising only a peripheral wall inclined at an angle of 50 to 90 °. 6. The centrifuge according to claim 4, wherein the peripheral slot has an inner peripheral wall inclined in a direction toward the central axis of the rotor at an angle of 50 to 90 ° with respect to a direction perpendicular to the central axis of the rotor. 7. The centrifuge according to claim 6, wherein the inner peripheral wall of the peripheral slot defines an arc or spiral with respect to the central axis of the rotor in an imaginary plane transverse to the central axis of the rotor. 8. A peripheral slot around the center axis of the rotor.
The centrifugal separator according to claim 4, which exists within 360 °. 9. A peripheral slot around the center axis of the rotor.
The centrifuge according to claim 4, wherein the centrifuge is present in a spiral shape at 360 ° or more without changing the angle in the direction of the central axis. 10. The centrifuge according to claim 4, wherein the peripheral slots are spirally arranged at an angle of 360 ° or more around the center axis of the rotor and inclined at an angle in the direction of the center axis. 11. The centrifuge according to claim 4, wherein the peripheral slot has a separation plate for animal cells in the slot.