JPH02200176A - Cell culture method and device - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a cell culturing method and a culturing device, and more particularly to a culturing method and culturing device for stably culturing cells at high density over a long period of time. It is something.

[Conventional technology]

Cell culture techniques are important for the production of viruses, vaccines, antivirals such as interferons, or biological drugs such as hormones.

Furthermore, in recent years, the production of monoclonal antibodies targeting specific protein awards has been achieved by culturing hybridomas made from antibody-producing cells and myeloma, and solving this technology is an industrially important theme.

Conventionally, cell culture has been carried out on a laboratory scale using culture bottles and the like. On the other hand, in recent years, several proposals have been made for industrial cell culture methods and apparatus for the purpose of producing the above-mentioned useful substances. These proposals are broadly classified into two methods: adherent culture and suspension culture, and the method is determined depending on the characteristics of the cells to be cultured.

The present invention relates to apparatus and methods for cell culture in suspension culture. Regarding methods of culturing cells by suspension culture, methods have been proposed in which a spinner flask is provided with a controlled agitation function, for example by a magnetine cooler or an impeller on a mechanically driven shaft. ing. However, in the above method, since the cells are cultured in a fixed amount of nutrients, the growth and proliferation of cells stops at a relatively low density. In such suspension culture of cells, in order to culture in large quantities and at high density, the culture is generally carried out while supplying new culture medium into the culture tank and discharging old culture medium containing growth-inhibiting substances to the outside of the culture tank. A method was proposed, and this method is called the perfusion culture method. What is important when using this method for irrigation is to efficiently separate the cells in suspension from the old culture medium over a long period of time, remove the old culture medium from the culture tank, and remove the old culture medium from the culture tank. The aim is to maintain the cell growth environment under optimal conditions.

Furthermore, what is industrially important for obtaining useful substances by cell culture is obtaining the target useful substances at high concentrations. Generally, in cell culture, the concentration of useful substances produced by cells in the culture medium is extremely low, and complicated means are required to separate and purify useful substances from the culture medium, which is one of the causes of reduced recovery rates and increased costs. It is one. For this reason, it is strongly desired to culture at the highest possible cell density.

Note that, for example, Japanese Patent Application Laid-Open No. 61-257181 is related to this type of device.

[Problem to be solved by the invention]

The above-mentioned conventional technology does not take into account stable separation of cells from a suspension culture solution over a long period of time, and has the problem that sufficient perfusion culture cannot be performed and high-density culture cannot be performed. That is, the animal cells and the culture solution can be separated as long as they can be separated while the cells are grown in a sterile environment in a manner that does not allow the cells to be mixed into the separated culture solution, and is usually carried out in a culture tank.

Separation means include a hollow fiber module, centrifugation, and a method in which a settling zone is provided in a culture tank. Among these methods, the method using a hollow fiber module has a large filtration area per volume, and the old culture solution is drained out of the culture tank through the membrane, and then the membrane is backwashed with fresh culture medium. It is suitable for perfusion culture because it supplies a medium. However, membrane separation methods always have the problem of clogging due to membrane contamination, which is particularly noticeable when separating animal cells, making it difficult to perform stable separation over a long period of time.

Therefore, an object of the present invention is to provide a method and apparatus that can perform stable perfusion culture over a long period of time and enable high-density culture.

[Means to solve the problem]

In order to achieve the above object, the cell culture method of the present invention involves culturing cells in suspension while partially discharging the culture solution from the suspension culture solution to the outside of the culture tank through a membrane provided in the culture tank. In a cell culture method in which continuous culture is performed while continuously supplying fresh medium, the flow direction of the culture solution to keep the cells in a suspended state in the culture tank is determined by adjusting the flow direction of the culture solution through the filtration membrane. time and
The purpose is to reverse the flow when fresh medium is supplied.

Furthermore, in the cell culture method of the present invention, the flow direction of the culture solution for keeping the cells in a suspended state in the culture tank is the direction in which the culture solution is stirred by the stirring means, and the culture solution is flowed through the filtration membrane. It is characterized in that the stirring direction is reversed between when discharging the medium and when supplying a fresh medium.

Furthermore, while culturing cells in a suspension state, the present invention allows part of the culture solution to be discharged from the suspension culture solution to the outside of the culture tank via a membrane provided in the culture tank, and then continuously supplies fresh culture medium. In a cell culture method in which continuous culture is performed while performing continuous culture, clogging of the membrane by cells is removed by restoring the deformation of the cells that occurs when the culture medium is discharged through the filtration membrane when fresh medium is supplied. The cell culture method is characterized by the following.

Furthermore, while culturing cells in a suspended state, the present invention allows part of the culture solution to be discharged from the suspension culture solution to the outside of the culture tank via a filter membrane provided in the culture tank, and then continues to supply fresh culture medium. In a cell culture apparatus that performs continuous culture while performing continuous culture, there is provided a stirring means for stirring the culture solution to suspend the cells, and a part of the culture solution to the outside of the culture tank through the filtration membrane. discharge, and
A culture solution discharge/supply means for supplying a fresh medium; a culture solution discharge/supply control means for controlling the culture solution discharge/supply by the culture solution discharge/supply means; and a culture solution discharge/supply control means for controlling the stirring direction of the stirring means to control the culture solution discharge.・Equipped with a stirring direction control means that controls in synchronization with the supply, the stirring direction of the culture solution by the stirring means is reversed between when the culture solution is discharged through the filtration membrane and when fresh culture medium is supplied. A cell culture device characterized in that it is configured to obtain a cell culture device.

Furthermore, in the cell culture apparatus of the present invention, the stirring means has stirring blades that can be rotated in forward and reverse directions, and the culture solution discharge/supply control means controls the opening/closing of the valve of the culture solution discharge/supply route and the operation of the pump. The stirring direction control means controls the stirring direction of the stirring means, and the stirring direction control means controls the stirrer that rotates the stirring blade and the operation of the stirrer in synchronization with the opening/closing of the valve of the culture liquid discharge/supply route and the operation of the pump. The cell culture apparatus comprises means for controlling the direction of rotation of the stirring blade to be reversed at a predetermined time.

Furthermore, while culturing cells in a suspended state, the present invention allows part of the culture solution to be discharged from the suspension culture solution to the outside of the culture tank via a filter membrane provided in the culture tank, and then continues to supply fresh culture medium. In a cell culture apparatus that performs continuous culture while performing continuous culture, there is provided a stirring means for stirring the culture solution to suspend the cells, and a part of the culture solution to the outside of the culture tank through the filtration membrane. discharge, and
A culture solution discharge/supply means for supplying a fresh medium; a culture solution discharge/supply control means for controlling the culture solution discharge/supply by the culture solution discharge/supply means; and a culture solution discharge/supply control means for controlling the stirring direction of the stirring means to control the culture solution discharge.・Equipped with a stirring direction control means that controls in synchronization with the supply, the stirring direction of the culture solution by the stirring means is reversed between when the culture solution is discharged through the filtration membrane and when fresh culture medium is supplied. and the culture solution discharge/supply means is comprised of a plurality of pairs of mechanisms that also serve as oxygen supply means, and the culture solution discharge/supply and oxygen supply are alternately performed for each of the plurality of pairs of mechanisms. The present invention provides a cell culture apparatus characterized by comprising a control means for controlling the cell culture.

As the above-mentioned filtration membrane, any membrane can be used as long as it is suitable for the purpose of the cell culture method of the present invention, and for example, a hollow membrane is preferably used.

Furthermore, as the fresh medium supplied through the above-mentioned filtration membrane, any medium suitable for growing the target cells can be used, but one without protein components is particularly suitable, It is preferable that the protein component is supplied to the culture tank without passing through a filtration membrane.

In the culture method of the present invention, the cells to be cultured may be of any type as long as they can grow in suspension, and include not only natural animal cells but also cells that have been modified artificially or by genetic manipulation, such as hybridomas. You can. The cells may also be lymphocyte-derived cells that produce lymphokines, or diploid cells that produce useful physiologically active substances such as interferon. Furthermore, it may be a cell that produces various monoclonal antibodies,
It may be a plant cell or a microscopic substance.

In the suspension cell culture according to the present invention, cells to be cultured are cultured in a culture tank in a suspended state in a culture solution. As described above, this cell suspension is achieved by reversing the flow direction of the culture solution at predetermined time intervals, and the reversal of the flow direction is adjusted in the culture tank using, for example, a magnetic cooler. This is done by stirring means. The culture solution is an aqueous medium consisting essentially of water, to which additive components commonly used in cell culture, such as various inorganic salts, vitamins, glucose, and amino acids, are added. Proteins such as animal serum, albumin, and insulin are essential for cell proliferation, and appropriate amounts of each are added.

As the cell separation membrane for performing perfusion culture in the present invention, a hollow fiber membrane is preferable because it can provide a large filtration area per volume. The membrane is a hydrophobic membrane made of Teflon or polyethylene,
Any hydrophilic membrane made of cellulose may be used. In addition, the pore size of the membrane is, for example, when the animal cell is a hybridoma, -
Since the size of a is IOpm or more, it is sufficient that it is 5 μm or less.

In carrying out the method of the present invention, 1. Although it is desirable to maintain the liquid level in the culture tank at a substantially constant level when supplying new culture medium and discharging old culture solution, this is not always necessary. The supply of new culture medium and the discharge of old culture solution can be performed independently and continuously, or can be performed intermittently.

In the culture method of the present invention, in order to maintain a constant oxygen concentration in the suspension, oxygen or an oxygen-containing gas may be directly supplied into the suspension.
Alternatively, other supply means may be used. Another supply method is, for example, a method in which oxygen is diffused and dissolved by flowing oxygen through a porous oxygen supply pipe immersed in a culture tank.

In order to perform culture efficiently, it is necessary to uniformly supply new culture solution and oxygen into the suspension, while draining the old culture solution out of the tank, so the suspension must be well stirred. It is desirable to be present.

As a stirring method, a mechanical stirring method such as rotation of a stirring blade is suitable for this purpose.

In addition, in culture, the ratio of supplying new culture solution to the effective culture volume of the culture tank (It conversion rate) is -0.2 to 10 per day.
．． Preferably, it is appropriate to set it in the range of 0.5 to 5.

The outline of the cell culture apparatus of the present invention is shown in FIG. 1, which includes a culture tank equipped with an enzyme supply tube 4, a hollow membrane 3, and a stirring blade 2, and an inverted Stark-5 installed externally corresponding to the stirring blade. Consisting of

FIG. 2 shows an example of the device configuration and control means necessary for changing the stirring direction of the culture solution according to the present invention. That is, a stirrer control box 6 that controls the rotation direction of the reversing Stark-5 and pumps P-1+P-2.
, P-3 and electric valves v-1 and V-2 are controlled by a timer 7 to perform perfusion culture.
The operating states of the electric valves V-1 and V-2 and the reverse Stark-5 over the course of the culture time are shown in FIG.

In addition to controlling the pump, electric valve, and reversing stirrer using a timer as described above, it is also possible to control the pump, electric valve, and reversing stirrer using a liquid level gauge (level sensor) provided in the culture tank.

FIG. 4 shows a combination of a cell separation membrane and an oxygen supply membrane. That is, during the filtration and backflow cycles in the hollow fiber membrane 3A, oxygen B is aerated through the hollow fiber membrane 3B, and oxygen is supplied to the culture tank 1. After the filtration and backflow cycle with the hollow fiber membrane 3A is completed, the filtration and backflow cycle is started with the hollow fiber membrane 3B, and oxygen B is aerated through the hollow fiber membrane 3A to supply oxygen to the culture tank 1. A porous Teflon membrane is suitable for such oxygen supply and cell separation. According to this method, the filtration load on the membrane is halved, and membrane cleaning is strengthened by oxygen aeration, so the filtration life of the membrane is significantly improved.

[For production]

The rotation of the stirrer used to stir the culture medium in order to keep animal cells suspended in the culture tank is now different between when the culture medium is filtered and discharged using the hollow fiber membrane and when the membrane is washed by supplying fresh medium. Operate. For this reason, cells deformed by stirring the culture medium during filtration enter some of the pores of the membrane and cause clogging, but when cleaning the membrane by supplying fresh medium, the direction of stirring of the culture medium is reversed, so the pores of the membrane become clogged. The shape of the cells that are deformed and clogged in the pores of the membrane is restored, so that they are easily separated from the pores and the membrane is thoroughly washed. Note that it is preferable that the membrane cleaning at this time be performed using a fresh medium that does not contain protein components. If this is done, the inner surface contamination of the hollow fiber membrane due to precipitation of protein components in the culture solution will be halved. Here, protein components essential for cell proliferation are separately supplied to the culture tank.

Further, in the cell culture device, a plurality of pairs of hollow fiber membranes are installed in a culture tank, and oxygen supply and filtration/membrane cleaning cycles are alternately performed via the plurality of pairs of hollow fiber membranes. The cells deform during filtration and clog the pores of the membrane. However, the membrane is sufficiently cleaned because the clogged cells are released from the pores of the membrane during membrane cleaning and especially when oxygen is supplied.

In the method and apparatus described above, means constituted by a timer and a control box or a liquid level sensor and a control box is used to rotate the stirring blades in opposite directions during filtration and membrane cleaning. The control box controls the rotating direction of the 0-reversing stirrer, which is electrically linked to the reversing stirrer, all valves and pumps, the opening and closing of the valves, and the operation of the pump to appropriately reciprocate in accordance with the cycles of filtration and membrane cleaning. send electrical signals.

Furthermore, the structure in which multiple pairs of hollow fiber membranes alternately operate in oxygen supply and filtration/cleaning cycles is possible because an oxygen concentration sensor is provided near at least one hollow fiber membrane. When the oxygen concentration increases (at this time, this hollow fiber membrane is supplying oxygen), the oxygen concentration sensor sends an electrical signal to the regulator located in the middle of the oxygen supply pipe, and this signal is sent to the regulator. The pumps and valves directly connected to the membranes are operated, and the hollow membranes are used for filtration and backwashing. Next, when the oxygen concentration near this hollow fiber membrane decreases,
At this time, the hollow fiber membrane near the oxygen concentration sensor, where an electrical signal opposite to that described above is sent from the oxygen concentration sensor to the regulator, is arranged to supply oxygen, and the cycle is repeated.

In addition, the oxygen concentration sensor and the regulator are electrically linked, and the regulators are linked to the pump and valve, respectively, so that the oxygen concentration in the culture tank is kept almost constant from beginning to end by a similar effect to that described above. dripping

Further, the unit is submerged in the suspension culture solution due to the hollow fiber membrane structure described above, and fresh culture medium is supplied from one end of the capillary bundle in the unit through the conduit, and the fresh medium is supplied through the hollow fiber membrane. A medium is supplied into the suspension culture, and
The old culture solution containing waste products, metabolic products, and other substances that inhibit the growth of cells produced by the cells passes through the hollow fiber membrane and flows out of the culture tank through the capillary bundle at the other end of the unit.
Cells can be easily cultured in large quantities and at high density in a suspension culture solution.

〔Example〕

Hereinafter, the present invention will be explained in detail using examples.

However, the present invention is not limited to these examples.

<Example 1> (1) Culture device A culture system shown in FIG. 1 was used. The culture tank 1 has a built-in Teflon hollow fiber membrane (2φ x 6m) 3 with a pore diameter of 2 μm, and one end of the module is provided with an outflow port for the culture solution and an inlet port A for the fresh medium, which can be switched by a valve. . In addition, the net volume of culture tank 1 is approximately 20001
B! It is.

(2) Culture solution Eagle's MEM medium and newborn calf serum were used.

(3) Culture method and results A culture medium (containing 10% newborn calf serum) was introduced into the above-mentioned culture tank, which had been sterilized by autoclaving in advance, so that the net culture volume was approximately 2,000 μm, and this was added to the strain derived from rat ascites hepatoma. cells were inoculated at 1 x 10 cells/-. In culture tank 1, oxygen gas B containing carbon dioxide (dissolved oxygen
ppm) through an oxygen supply pipe 4 made of porous polytetrafluoroethylene under automatic control. The culture solution in the culture tank is maintained at 37°C. A marine type stirring blade 2 was installed in the culture tank, and the stirring speed was 60 rpm. Incidentally, the stirring blades 2 are interlocked with a magnetic reversing stirrer 5 which can be reversed for an arbitrary period of time using a timer. The culture is as follows■,■
, ■.

■ Filtration process 200% of the culture solution is passed through the hollow fiber membrane 3 by the pump P-2.
Filter at mt/h for 2.5 hours and drain the old culture solution.

The reversing stirrer 5 was rotated clockwise.

■Backwashing process pump P-1 pumps serum-free fresh medium A 200-/h.
The hollow fiber membrane 3 was backwashed for 2.5 hours, and serum C was separately pumped into the culture tank L at 20 m/h for 2.5 hours using pump P-3 to supply fresh medium and serum. do.

The reversing stirrer 5 was rotated to the left.

■Pause process All pumps P-1, P-2, and P-3 are stopped for one hour.

The reversing stirrer 5 was rotated clockwise.

The above perfusion culture operation was repeated for 40 days, with one cycle (6 hours) of ■→■→■.

As shown in Table 1, the cell density and survival rate were both extremely good.

(Margins below this page) Inversion (add protein separately) (fi 艮J Inversion (contain protein in medium) (艮) <Example 2> The same culture apparatus and culture solution as in Example 1 were used. Experimental method The ■backwashing process in Example 1 was performed using pump P-1.
Fresh serum-containing (10χ) medium was grown at 200 m/h for 2 hours.
．． The hollow fiber membrane 3 is backwashed for 5 hours (serum supply by pump P-3 is stopped). At this time, the reversal start-5
was rotated to the left. Everything else was the same except for these. As shown in Table 2, both cell density and survival rate were good.

(Margin below the essence) <Example 3> The same culture device and culture solution as in Example 1 were used. The cells to be cultured were the established cell line HeLa.

The experimental method was to perform the backwashing process in Example 1 using pump P-
1, the hollow fiber membrane 3 is backwashed with a serum-containing (10x) fresh medium at 200-/h for 2.5 hours (serum supply by pump P-3 is stopped). At this time, the reverse start
5 was a left rotation. Everything else was the same except for these. As shown in Table 3, both cell density and survival rate were good.

(Leaving space below the essence) Inverted table (medium contains protein) (good) Cells were HeLa <Example 4> (1) Culture device The culture system shown in FIG. 3 was used. Culture tank 1 was equipped with a hollow fiber membrane made of polytetrafluoroethylene with a pore diameter of 2 μm (
2φ x 6m) 3A and 3B are built-in, and a pipe that doubles as an oxygen supply pipe and a filtration backwash immersion pipe is buried in each. One end of the module is provided with a culture solution outflow port and a fresh culture medium inflow port A, which are switched by a valve.

In addition, the net volume of the culture tank 1 is about 1800-.

(2) Culture solution Eagle's MEM medium and newborn calf serum were used.

(3) Culture method and results A culture medium (containing 10% newborn calf serum) was introduced into the culture tank, which had been sterilized by autoclaving in advance, so that the net culture volume was about 1800 ml, and a strain derived from rat ascites liver cancer was introduced. Cells were inoculated at 1×10 b cells/d.

Into the culture tank 1, oxygen gas B containing carbon dioxide gas (so that dissolved oxygen is 3 ppm) is automatically controlled and fed through an oxygen supply pipe 4 made of porous polytetrafluoroethylene. The culture solution in the culture tank is maintained at 37°C. A marine type stirring blade 2 was installed in the culture tank, and the stirring speed was 60 rpm. Cultivation was carried out as described below.

(2) Filtration and backwashing process Pump P-2: P-1 filters and backwashes the culture solution through the hollow fiber membrane 3A at 200-/h for 5 hours, and discharges the old culture solution. During this time, oxygen gas B is introduced into the hollow fiber membrane 3B. Star 5-5 is clockwise rotation. Pump P-3 pumps serum C into culture tank 1 at 20 d/h for 2.5 hours to supply fresh medium and serum.

The reversing stirrer 5 was rotated counterclockwise.

■Pause process Pump P-1, P~2. Stop all P-3 for 1 hour.

Star 5-5 is clockwise rotation.

The perfusion culture operation was repeated for 40 days, with one cycle (6 hours) of ■→■ as described above. The result is the fourth
As shown in the table, both cell density and survival rate were good.

Table 1 (Medium contains protein) (Good) Double hollow fiber membrane <Example 5> The same culture device and culture solution as in Example 4 were used. The cells to be cultured were HeLa, which is a type of established cell line.

The experimental method was to repeat the filtration and backwashing process in Example 4 by pumping 1 serving of serum-containing (10χ) fresh medium using pump P-2:P-1.
The hollow fiber membrane 3-A is filtered and backwashed at 50 d/h for 5 hours. (The supply of serum by pump P-3 was stopped.) At this time, the inversion Starcy was rotated to the left. Everything else was the same except for these. As shown in Table 5, both cell density and survival rate were good.

(Left below the essence) (Contains protein in medium) (Good) Double hollow fiber membrane Cells are HeLa <Comparative Example 1> The same culture device and culture solution as in Example 1 were used. The experimental method was the same as in Example 1 except that the rotating direction of the inverted Starshi-5 was always clockwise. The result is the 6th
As shown in the table.

(Margins below essence) Constant clockwise rotation (no protein included) (good) <Comparative Example 2> The same culture device and culture solution as in Example 4 were used. The experimental method was as follows: The rotating direction of the inverted Sturge-5 in Example 1 was always clockwise, and the backwashing process was performed by pumping P-1 with serum-containing (10χ) fresh medium at 200 m//h for 2.5 m/h.
The hollow fiber membrane 3 is backwashed for an hour (serum supply by pump P-3 is stopped). Everything else was the same as in Example 1. The results are shown in Table 7.

(Leaving space below the essence) Table 7 Constant clockwise rotation (contains protein) (defective) Double hollow fiber membrane From the above examples and comparative examples, when clogging of the hollow fiber membrane occurs,
Due to the increased pressure loss, filtration of the culture solution through the membrane and backwashing with fresh culture medium cannot be performed sufficiently, and the replacement rate gradually decreases. As a result, old culture medium cannot be removed and fresh culture medium cannot be supplied, resulting in insufficient supply of nutrients (e.g. glucose and amino acids) and accumulation of growth-inhibiting factors (e.g. cell waste components such as lactic acid and ammonia). The proliferation reaches a plateau and the survival rate decreases. Than this,
It can be seen that it is important to prevent a decrease in the replacement rate due to clogging of the hollow fiber membrane.

According to this example, in the backwashing of hollow fiber membranes, by (1) washing with a serum-incompatible fresh medium and (2) reversing the stirring direction of the culture solution, it is possible to reduce the internal contamination of the hollow fiber membranes by protein components and to reduce the contamination of the hollow fiber membranes during filtration. Since detachment occurs due to restoration of the shape of the cells that clog the pores, this has the effect of significantly reducing clogging of the hollow fiber membrane.

From the above, although the culture experiment was conducted with the substitution rate set at 1.0, it is clear that if the substitution rate is increased, the cell density will further increase.

〔Effect of the invention〕

The structure described above has established a method of culturing cells at high density and with a high survival rate over a long period of time using suspension culture (suspension culture) or perfusion culture in a culture medium, and also provides equipment for this purpose. It became possible to do so.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing a cell culture device of the present invention, FIG. 2 is a diagram showing a cell culture device equipped with a control means of the present invention, and FIG. 3 is a diagram showing a cell culture device equipped with a plurality of pairs of hollow fiber membranes. FIG. 4 is a diagram showing a cell culture device, and FIG. 4 is an operational diagram showing the rotation direction of the inverted stargie, pump operation, and valve opening/closing in the cell culture device of the present invention, with the horizontal axis as the time axis. l... Culture tank, 2... Stirring blade, 3 (3A, 3B
)...Hollow fiber membrane, 4...Oxygen supply tube, 5...Reversing stirrer 6...Control box, 7...Timer, 8...Protein component introduction tube, 9...Oxygen Concentration sensor 10...Adjuster, 11...Alkaline tank, 12...Solenoid valve for pH control, 13...
・pH electrode, work 4...pH electrode connector, 15...
Tachometer, 16... Rotation speed adjustment handle, 17... Bearing, 18... Peephole, 19... Heater, 20...
・Thermosensor, 2 pieces...Temperature controller, 50...
- Stirrer A... Fresh medium inlet, B... Oxygen gas module, C... Protein component module,
D... Culture solution outlet.

Claims (1)

[Claims] 1. Partial discharge of the culture solution from the suspension culture solution to the outside of the culture tank through a membrane provided in the culture tank while culturing cells in a suspended state;
In a cell culture method in which continuous culture is performed while continuously supplying fresh medium, the flow direction of the culture solution to keep the cells in a suspended state in the culture tank is determined by adjusting the flow direction of the culture solution through the filtration membrane. A cell culture method characterized by reversing the time and supplying a fresh medium. 2. The flow direction of the culture solution to keep the cells in a suspended state in the culture tank is the direction in which the culture solution is stirred by the stirring means, and when the culture solution is discharged through the filtration membrane and when fresh culture medium is supplied. 2. The cell culture method according to claim 1, wherein the stirring direction is reversed from time to time. 3. Partial discharge of the culture solution from the suspension culture solution to the outside of the culture tank via a membrane provided in the culture tank while culturing cells in a suspended state;
In a cell culture method in which continuous culture is performed while continuously supplying fresh medium, the deformation of the cells that occurs when the culture medium is drained through the filtration membrane is restored when fresh medium is supplied. A cell culture method characterized by removing membrane clogging caused by cells. 4. Claim 1, wherein the filtration membrane is a hollow fiber membrane.
4. The cell culture method according to any one of 3 to 3. 5. The cell culture method according to any one of claims 1 to 4, characterized in that the fresh medium supplied through the filtration membrane is a medium made of components excluding protein components, and clogging of the filtration membrane is removed. 6. While culturing the cells in suspension, part of the culture solution from the suspension culture solution is discharged to the outside of the culture tank via a filtration membrane installed in the culture tank, and then continuous culture is performed while supplying fresh medium. In a cell culture apparatus that performs the following steps, there is provided a stirring means for stirring the culture solution to suspend the cells, a part of the culture solution is discharged to the outside of the culture tank through the filtration membrane, and a fresh medium. a culture solution discharge/supply means for controlling the culture solution discharge/supply by the culture solution discharge/supply means; and a culture solution discharge/supply control means for controlling the culture solution discharge/supply by the culture solution discharge/supply means; and stirring direction control means for controlling the stirring direction synchronously with the
1. A cell culture device characterized in that the direction of agitation of a culture medium by a stirring means can be reversed between when the culture medium is discharged through a filtration membrane and when a fresh medium is supplied. 7. The stirring means has stirring blades that can be rotated in forward and reverse directions, and the culture solution discharge/supply control means includes means for controlling the opening/closing of the valve of the culture solution discharge/supply route and the operation of the pump; A stirring direction control means for controlling the direction of the stirring blade rotates the stirring blade by controlling the stirrer that rotates the stirring blade and the operation of the stirrer in synchronization with the opening/closing of the valve of the culture solution discharge/supply route and the operation of the pump. 6. The cell culture device according to claim 5, further comprising means for reversing the direction at a predetermined time. 8. Claim 6, characterized in that the filtration membrane is a hollow fiber membrane.
Or the cell culture device according to claim 7. 9. Any one of claims 5 to 7, characterized in that the fresh medium supplied through the filtration membrane is a medium consisting of components excluding protein components, and the liquid containing the protein components is supplied directly into the culture tank. 1. The cell culture device according to item 1. 10. While culturing cells in suspension, part of the culture solution from the suspension culture solution is drained out of the culture tank through a filter membrane provided in the culture tank, and then fresh culture medium is continuously supplied. In a cell culture device that performs continuous culture while stirring, a stirring means for stirring the culture solution to suspend the cells, a part of the culture solution to be discharged outside the culture tank through the filtration membrane, and , a culture solution discharge/supply means for supplying fresh culture medium, and a culture solution discharge/supply means for supplying fresh culture medium;
a culture solution discharge/supply control means for controlling the culture solution discharge/supply by the supply means; a stirring direction control means for controlling the stirring direction of the stirring means synchronously with the culture solution discharge/supply;
The stirring direction of the culture solution by the stirring means can be reversed between when the culture solution is discharged through the filtration membrane and when fresh culture medium is supplied, and the culture solution is discharged and supplied. The means consists of multiple pairs of mechanisms that also serve as oxygen supply means,
A cell culture apparatus characterized in that each of the plurality of pairs of mechanisms is provided with a control means for alternately discharging and supplying the culture medium and supplying oxygen.