JPH02138963A - Cell culture method and device - Google Patents

Abstract

PURPOSE:To suppress liquid flow so as not to increase the number of cells moving from the first to the second zones from that of the cells increasing in the first zone and carry out supply of nutrient sources and removal of waste materials for a long period by moving the nutrient sources and waste materials through a partition wall. CONSTITUTION:An apparatus is composed of an inner vessel part 102, divided with a cylindrical partition wall 104 having the closed bottom and suspending cells, an outer vessel part 103 for flowing a liquid and a jacket 105 for circulating warm water for keeping the temperature of the culture vessel 101 constant. The number of cells moving from the inner vessel 102 through the partition wall 104 to the outer vessel 103 is reduced. The liquid is fed from a culture medium feed pipe 106 to the outer vessel 103 with a pump 108 and taken out of the outer vessel part 103 through a taking out pipe 107 with a pump 109. The flow rates of the pumps 108 and 109 are changed manually or using a control computer.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application of the Invention] The present invention relates to cell culture, and particularly to a cell culture method suitable for high-density, large-scale culture of cells grown in suspension, and a culture device therefor.

[Conventional technology]

Mass culture of animal cells is an important technology as a means of producing physiologically active substances used in pharmaceuticals and diagnostic agents.

Animal cells are broadly divided into those that can grow in suspension and those that grow only when attached to solid surfaces. As devices that enable the former type of floating culture, a spinner flask, a rotor bottle, or a mechanically stirred culture tank is used. However, in culture using the above device,
Since cells are cultured in a fixed amount of nutrient source, in many cell lines only a cell concentration of about 1×1, O'cel], s/m Q can be obtained. As a method for culturing to an even higher cell concentration, a method is known in which the cultured cells are aseptically separated from the culture solution by centrifugation, collected, resuspended in a fresh medium, and cultured. That is, a high cell concentration can be obtained by removing waste products excreted by the cells and accumulated in the culture solution, and adding a fresh medium to supply a nutrient source. However, repeating operations such as centrifugation tends to invite the invasion of various bacteria into the culture system, and the operations are complicated. Therefore, a method that continuously supplies nutrients to the culture system and removes waste products from the culture system is desirable.

In order to achieve this continuous supply of nutrients and removal of waste products, a cell separation method that does not damage cells and is stable over a long period of time is required. In recent years, several methods have been proposed for high-density, mass-culture of cells that continuously supply nutrients and remove waste products. For example, there is a method and apparatus in which a cylindrical filter having a surface parallel to the rotation axis is rotated to replace the culture solution through filtration while preventing clogging of the filter by centrifugal force (US Pat. No. 3,647,632). . There is also a method and apparatus in which a tube for sedimentation of cells is provided in the culture tank with an opening at the bottom of the tank, and a fresh medium is added while draining the supernatant obtained as a result of sedimentation of the cells to the outside of the culture tank (
(Special Publication No. 61-36915). Alternatively, a semipermeable membrane with specific permeability for selected nutrients, waste products, or gases may be used to separate the cell-containing culture medium and the cell-free liquid on both sides of the semipermeable membrane. There is a method and apparatus for culturing cells by supplying nutrients, waste products, or gases to a culture solution containing cells by flowing them in parallel, and by diffusion through the semipermeable membrane, or by discharging them from the culture solution (Japanese Patent Application Laid-Open No. 1983-1999). 17
No. 5877, Japanese Unexamined Patent Publication No. 5917E) and No. 878).

[Problem to be solved by the invention]

However, cell separation methods using filters tend to become clogged when used for a long period of time, so it is necessary to devise ways to prevent this, but there is still no method that can withstand industrial-level use. Furthermore, in the method using a sedimentation tube, cells must be left stationary in the sedimentation tube for a long time, resulting in the problem of cell damage due to oxygen deficiency. In addition, selected nutritional sources,
In methods using semipermeable membranes that have specific permeability for waste products or gases, the pore size of the membrane is very small to allow selective permeation of molecules, and the diffusion rate of nutrients or waste products through the membrane is low. . Therefore, it is difficult to obtain a sufficient amount of diffusion of these substances during high-density, large-scale culture. Furthermore, the semipermeable membrane has low mechanical strength and is not suitable for long-term culture.

In view of the above problems, an object of the present invention is to provide a system that does not damage cells, can stably supply nutrients and remove waste products over a long period of time, and is suitable for high-density, large-scale culture of cells that can grow in suspension. An object of the present invention is to provide a culture method and device.

[Means to solve the problem]

The means for achieving the above objective will be described in detail below.

The first feature of the present invention is that the partition wall has holes with a diameter that allows cells to pass through, so that a first region in which a culture solution with suspended cells exists and a second region in which a solution containing a physiological buffer or a nutrient source exists. and moving at least one of a nutrient source, a waste product, or a product between the first region and the second region via the partition wall, and further moving from the first region to the second region via the partition wall. A method for culturing cells that suppresses liquid flow through a partition wall so that the number of cells does not exceed the number of cells that increases due to proliferation in the first region.

A second feature of the present invention is that the partition wall has holes with a diameter that allows cells to pass through, so that a first region in which a culture solution with suspended cells exists and a second region in which a solution containing a physiological buffer or a nutrient source exists. means for moving at least one of a nutrient source, a waste product, or a product between the first region and the second region via a partition wall;
The cell culturing device has means for suppressing liquid flow through a partition wall so that the number of cells migrating from the region to the second region does not exceed the number of cells increasing in the first region due to proliferation.

a nutrient source between the first region and the second region via the partition;
Transfer of waste products or products can be achieved by adding a medium to the first region and/or the second region and withdrawing the medium from the second region.

Here, if the number of cells migrating from the first region to the second region via the septum does not exceed the number of cells increasing in the first region due to proliferation, the number of cells in the first region increases. However, in order to perform efficient culture, it is desirable to reduce the number of migrating cells as much as possible. The movement of the cells is due to liquid flow through the septum. Therefore, when adding a medium to the first region, by controlling the addition flow rate and the flow rate of the medium passing through the septum per unit area, the number of cells that move from the first region to the second region through the septum can be controlled. can be suppressed. In addition, when adding the culture medium to the second region, the pressure difference between the first region and the second region through the partition wall is made substantially zero, thereby suppressing the liquid flow through the partition wall, and adding the medium through the partition wall. The number of cells migrating from one region to the second region can be suppressed.

The pressure difference between the first region and the second region through the partition wall can be made substantially zero by making the pressures of the gas phase portion of the first region equal to the pressure of the gas phase portion of the second region. Here, when adding a medium to the second region, the movement of substances through the partition wall is mainly due to diffusion. The driving force of this diffusion is the concentration difference between the substances in the first region and the second region.

If the medium is added to the first region using a septum that has pores that allow cells to pass through, the septum is less likely to become clogged than when a septum that has pores that are small in diameter is used. The required backwashing is also largely unnecessary for several weeks of culture. Therefore, the device configuration becomes simple and operation becomes extremely easy. In addition, during culture for several months, clogging may occur even when using a septum with holes large enough for cells to pass through. Short-term backwashing with liquid flow.

Alternatively, the obstruction can be easily recovered by forcibly extruding the obstruction for a short period of time by a liquid flow from the first region to the second region through the partition wall. For example, a backwash operation involves rapidly adding a culture medium to a second region, and causing a short period of liquid flow from the second region to the first region through a partition wall due to the hydrostatic pressure difference generated between the first and second regions. By creating a flow, it can be easily done without any special equipment. In addition, the forced extrusion operation of the occluded material is performed by rapidly adding the culture medium to the first region, and moving from the first region to the second region via the partition wall due to the hydrostatic pressure difference generated between the first and second regions. This can be easily done without special equipment by creating a short-term liquid flow. Furthermore, if a partition wall having pores with a diameter that allows cells to pass through is used, cell debris and polymeric waste products excreted by cells can easily pass through, thereby avoiding inhibition of growth. Next, when adding the medium to the second region using a septum having a diameter that allows cells to pass through, it is easier to add the medium to the second region through the septum than when using a septum having small diameter pores, for example, a semipermeable membrane. The high rate of movement of these substances facilitates high-density, large-scale culture.

Due to the features of the present invention described above, it is possible to easily maintain culture conditions favorable to cells, and it is also possible to create a compact device, thereby facilitating high-density, large-scale culture of cells.

Hereinafter, the present invention will be specifically explained with reference to the drawings.

FIG. 1 is a schematic diagram showing an example of the culture apparatus of the present invention.

In this FIG. 1, a cylindrical bulkhead 10 with a closed bottom is shown.
4, an inner tank part 102 in which the cells separated by 4 are suspended, an outer tank part 103 in which the liquid flows, and a jacket 105 in which hot water is circulated to keep the temperature of the culture tank 101 constant.
The device is configured by: By forming the partition wall 104 into a cylindrical shape with a closed bottom, there is no dead zone of stirring, and mild stirring conditions can be selected.

The partition wall 104 is composed of a woven fabric material (trade name: SUS fiber filter 5F-05) made by heating and pressing 5US316L fibers and molding them into a cylindrical shape, and the pore diameter thereof is nominally 5μ.
It is m. However, the material constituting the partition wall 104 is not particularly limited to the above-mentioned woven material, and may be any material as long as it does not inhibit cell proliferation. However, it is desirable that cells are difficult to adhere to. For example, polytetrafluoroethylene, ceramics, etc. can also be used. Further, the partition wall 104 can also be made of a material having an electrostatic charge of the same polarity as the electrostatic charge held by the cells in the inner tank portion 102. When this material is used, it is difficult for cells to approach the partition wall 104 due to electrical repulsion, and the number of cells that migrate from the inner tank section 102 to the outer tank section 103 via the partition wall 104 can be reduced. These materials include olefin polymers with ionic groups added to their main chains.

Furthermore, by applying a potential of the same polarity to the partition wall 104 as the electrostatic charge held by the cells in the inner tank 102, the same electrical repulsion effect as described above can be obtained. In this case, the partition wall 104
The applied voltage is preferably a weak voltage so as not to cause electrolysis of the liquid. Furthermore, it is desirable to use a partition wall 104 with a large number of holes in order to reduce the actual medium flow rate within the holes as much as possible. It is more preferable to use partition walls made of a woven material or a porous material. Further, it is desirable that the partition wall 104 has good moldability and high mechanical strength. This is because the partition wall can be freely configured according to the capacity and shape of the culture device, and it can withstand long-term culture. Further, the optimum pore diameter of the partition wall 104 is determined depending on the type of cell and the properties of the partition material.

From the culture medium supply pipe 124 to the inner tank section 10 using the pump 108
2 and/or from the culture medium supply pipe 106 to the outer tank section 103
The liquid is supplied to the extraction pipe 107 using the pump 109.
The liquid is extracted from the outer tank part 103. these pumps 1
08.109 flow rate can be changed manually or by instructions from the control computer.

By controlling the flow rates of the pumps 108 and 109 while monitoring the inner tank liquid level using the level sensor 121, the inner tank liquid level can be maintained. When the pump 108 is used to supply the liquid to the inner tank part 102 and the pump 109 is used to extract the liquid from the outer tank part 103, if the partition wall becomes clogged and the medium flow rate that can pass through the partition wall decreases, the inner tank part The liquid level rises.

When this liquid level rise is detected by the level sensor 121, if the pump 108 is stopped and the pump 109 is reversed to quickly add a predetermined amount of culture medium to the second area, the static The water pressure difference causes a liquid flow from the second region to the first region through the partition wall, allowing backwashing of the partition wall. After the backwashing operation, if the pump 109 is used to draw out the liquid from the outer tank section 103 and return it to the normal inner tank liquid level, it is possible to return to the normal liquid level control using the pumps 108 and 109. .

The liquid supplied from the medium supply pipe 106 includes physiological buffer,
A culture medium etc. can be used. Preferably, a medium is used. Furthermore, the liquid supplied from the supply pipe IQ6 may be a fresh culture medium or a mixture of the culture medium extracted from the outer tank 103 from the extraction tube ]-07 and a fresh culture medium in an appropriate ratio. . A culture medium can be used as the liquid supplied from the culture medium supply pipe 124. In addition, fresh medium or extraction tube 1.
A liquid obtained by mixing the culture medium extracted from the outer tank 103 with a fresh culture medium in an appropriate ratio can be used.

A stirring shaft 112 is immediately moved by a motor 111 on the central axis of the inner tank part 102, and a stirring blade 113 is located below the stirring shaft 112.
is provided. The method of stirring the liquid in the inner tank part 102 is not particularly limited to a mechanical stirring method using rotation of the stirring blade 113 attached to the stirring shaft 112, but a stirring method in which air is blown into the liquid in the inner tank part 102, etc. is used. You can also do that. However, if serum is added to the medium, it is desirable to select a method that does not cause foaming. Furthermore, when a mechanical stirring method is used, the shape of the stirring blades 113 is not particularly limited to the modified blade as shown in FIG. 1, and may be paddle-shaped, paddle-shaped, ribbon-shaped, etc.

However, since cells are relatively weak against the shear force caused by stirring, it is desirable to use blades with good stirring efficiency even at low speed rotation.

The rotation speed of the stirring shaft] 12 is preferably at least a speed at which the cells are sufficiently uniformly dispersed.

The method of supplying oxygen to the culture solution in the inner tank section 102 is as follows:
The ring sparger 114 can be used, for example, although it cannot be specified unconditionally because it is selected depending on the shape of the sparger 1, the amount of liquid in the inner tank part 102 and the outer tank part 103, the foamability of the liquid, etc. The oxygen-containing gas is led to the ring sparger 114 through the air supply pipe 116, and is then introduced into the inner tank 1 through the nozzle 115.
02 Sprayed on the liquid surface. Molecular oxygen moves from the air-liquid interface in the depression formed on the liquid surface, and oxygen is supplied to the culture solution in the inner tank section 102. The dissolved oxygen concentration of the culture solution in the inner tank section 102 can be controlled by changing the flow rate of the gas blown from the ring sparger 114, the partial pressure of oxygen in the gas, the rotational speed of the motor 111, etc. As another oxygen supply method, a sub-liquid aeration method may be used in which a porous sparger is provided in the liquid to exchange gas in the upper gas phase portion of the inner tank 102. In addition, the pressure equalizing port 120 provided in the bulkhead flange 19
It is possible to maintain the pressure difference between the gas phase portion of No. 2 and the gas phase portion of the outer tank portion 103 at substantially zero.

The pH control method of the culture solution is selected depending on the shape of the culture tank 101, the volume of the culture solution, etc., and cannot be determined unconditionally.
02 A method can be used that changes the carbon dioxide concentration in the gas blown onto the liquid surface. In addition, a method of appropriately adding an alkali or acid can be used.

A level sensor 121, a dissolved oxygen concentration sensor 122, a pH sensor 123, and a temperature sensor (not shown) are installed in the inner tank m1.02. A sterilization filter 118 is attached to the air supply pipe 116 and the exhaust pipe 117. The sterilizing filter 118 removes germs from the oxygen-containing gas and prevents germs from entering through the exhaust pipe 117.

FIG. 2 is a schematic diagram showing an example of the culture control system of the present invention. In FIG. 2, a culture device 101, an inner tank portion 102 for suspending the cells of the culture device 101 separated by a cylindrical partition wall 104 with a closed bottom, an outer tank portion 103 for circulating liquid, and an inner tank portion are shown. ], a tank 201 that stores the liquid to be supplied to o2 and/or the outer tank section 103, a tank 202 that stores the liquid recovered from the outer tank section 103, and a tank 202 that stores the liquid recovered from the outer tank section 103, and a tank 201 that is sent from the tank 201 to the inner tank section 102 and/or the outer tank section 103. Liquid pump 108, from the outer tank part 103 to the tank 202
pump 109, constant temperature water tank 203, constant temperature water tank 2
A pump 204 that sends hot water from 03 to the jacket 105 of the culture device 101, and an air mass flow controller 20.
5. Oxygen mass flow controller 206, carbon dioxide mass flow controller 207, level sensor 121 inserted into inner tank 102, dissolved oxygen concentration sensor 1
22, a pH sensor 123, a temperature sensor (not shown), a motor 111, and a control computer 208.

The control computer 208 controls each of the above sensors 1-21.1.
22 and 123, and based on this data, 0N10FF of the hot water pump 204, stepwise opening/closing operations of the mass flow controllers 205, 206, and 207, and rotation speed control of the motor 111 are performed. The control computer 208 also uses data input from the keyboard 209 such as the cell concentration in the culture solution in the inner tank section 102 and the concentration of nutrients and waste products in the solutions in the inner tank section 102 and the outer tank section 103. Inner tank part 102 and/or outer tank part 1
Decide the flow rate of the liquid to be added to the liquid feed pump 108.1.
Controls 09.

FIG. 3 is a schematic diagram showing another example of the culture apparatus of the present invention. In FIG. 3, a culture tank section 301 and a tube interior 3 are separated by a partition wall 303 formed into a hollow tube shape.
Consists of 02. Cultivating cells in the culture tank section 301,
A liquid is allowed to flow through the inside of the tube 302 . The partition wall 303 is installed in the culture tank section 301 in a spiral shape. By configuring the partition wall 303 in this manner, the surface area of the partition wall 303 can be increased.

FIG. 4 is a schematic diagram showing another example of the culture apparatus of the present invention. In FIG. 4, it is composed of a culture tank section 401 and an outer tank section 402, which are separated by a plate-shaped partition wall 403. Cells are cultured in a culture tank section 401, and a liquid is passed through an outer tank section 402.

The partition wall 403 has a horizontal cross section of 2 in the culture tank section 4-01.
They are placed together to form two rectangles. However, the outer tank part 402 does not have to be two areas in particular, but one area.
There may be one or three or more. In this way, the partition wall 4
03, the surface area of the partition wall 403 can be increased.

[Effect]

In the present invention, two regions are separated using a partition wall having pores large enough to allow cells to pass through, and cell proliferation is achieved by continuously adding a medium to the first region and/or the second region. Since inhibiting substances can be removed, high-density mass culture of cells grown in suspension becomes easily possible. However, it is necessary to prevent cell migration through the septum from becoming too large. This means that the number of cells migrating from the first region to the second region via the septum is the first. This is because when the number of cells in the area exceeds the increase in the number of cells due to proliferation, the number of cells in the culture tube 1 area decreases and eventually there are no cells, a so-called washout.

Example 1 Cells were cultured using a twin tube tapping flask.

The twin-tube tapping flask is a tapping culture method developed by Takaoka et al.; in vitro (InVjtro, 15 (
12), 949 (1979) were used, and were processed so that a partition material could be sandwiched between them.

A first tank and a second tank are separated by a partition wall. The inside of the tank is stirred by moving the stirrer up and down with a starter.

In each twin-tube tapping flask with a capacity of 200 mQ,
5US316L with 5μm pore diameter (nominal) and 400μm thickness
Woven cloth material (Tokyo Seimi SUS fiber filter 5
F-05) with an effective area of 2.1 cm2 was installed. DM-160AU medium (10% newborn calf serum,
80 mQ of 0.3 g/Q glutamine and 0.1 g IQ kanamycin supplemented with 2.8 g glucose (reinforced with glucose) were added to both the first and second tanks, and JTC1 strain cells were inoculated into the first tank. The cell concentration at this time is 5.9X 10'cells
/m Q. The stirring bar rotation speed was set at 40 rpm, and the culture temperature was set at 37°C.

The medium was added to the first tank at a flow rate of 160 mQ/day, and extracted from the second tank at a flow rate of 160 mQ/day. Table 1 shows the results of measuring cell concentrations in the first and second tanks on the first and second day after inoculation.

Table 1 From Table 1, the cell concentration in the first tank was almost constant. This is because the number of cells migrating from the first tank to the second tank via the septum was approximately equal to the number of cells increasing in the first tank due to proliferation. At this time, the flow rate of the medium passing through the partition wall per unit area was 75 mQ/Cm2°day.

Here, the cell size is approximately 10 μm, and 5US316L
Although the nominal pore diameter of the woven cloth-like material is larger than 5 μm, since the cells are deformable, if the flow rate of the medium passing through the septum per unit area is large, it is considered that the cells deform and pass through the septum.

From the above, when culturing JTC-1 cell lines using SUS 316 L woven fabric-like septums with a pore size of 5 μm, if the flow rate of the medium passing through the septum per unit area is set to 75 mu/am”・day or less, the first tank It was found that it is possible to increase the number of cells.

Example 2 Using the apparatus shown in FIG. 1, cells were cultured while adding a medium to the inner tank portion (corresponding to the first region) from the medium supply pipe 124.

The above 5US316L with a closed cylindrical wall at the bottom of the total volume 3, Op in a glass culture tank with a total volume of 8.0Q
A partition made of a woven cloth-like material was provided as shown in FIG.

A modified stirring blade immersed in the inner tank was rotated by a motor from above. A magnetic stirrer was placed in the bottom of the outer tank and rotated by Starcy. The stirring blade rotation speed was set to 1100 pp, and the magnetic stirrer rotation speed was set to 180 rpm.

Using the same medium as in Example 1, the inner tank medium volume was 2.5Q,
A medium was added so that the outer tank medium volume was 2.7Ω. The effective area of the partition wall at this time is 800 cm2. When JTC-1 strain cells were inoculated into the inner tank, 1,2 x 10
6 cells/mQ.

A culture medium was added to the inner tank using a peristaltic pump, and the culture medium was extracted from the outer tank while keeping the liquid level constant. The flow rate of the medium added to the inner tank is 2.7-6.3 mQ/cm2・d when expressed as the flow rate of the medium passing through the partition wall per unit area.
It is ay. Furthermore, the septum was not backwashed even once during the entire culture period.

The culture temperature is 37°C. Dissolved oxygen concentration and pH were controlled by spraying on the liquid surface using a sparger, and the flow rates of air, oxygen, and carbon dioxide were controlled so that the dissolved oxygen concentration was 2 Ppm and the pH was 7.2.

Changes in cell concentration in the inner tank and outer tank over time are shown in FIG. 5 by solid lines and dashed-dotted lines, respectively. The cell concentration in the inner tank reached 2.5 x 107 cells/mQ on the 15th day after the start of culture, the cell survival rate at that time was 91-%, and the cell concentration in the outer tank reached 8.5 x 1.04 cells/mQ. s/m Q. Since the cell concentration in the outer tank is 1/300 of that in the inner tank, the number of cells passing through the partition and flowing out of the culture system is within a negligible range.

From this, it was confirmed that high-density, large-scale culture of cells can be easily achieved using the culture tank of the present invention.

Example 3 Using the apparatus shown in FIG. 1, cells were cultured while adding a medium to the inner tank part (corresponding to the first region) from the medium supply pipe 124,
A test was conducted on clogging and recovery by backwashing in the 5US316L woven fabric material.

The above 5US316L with a closed cylindrical wall at the bottom of a total volume of 0.7Q in a glass culture tank with a total volume of 1.7Ω
A partition made of a woven cloth-like material was provided as shown in FIG.

A modified stirring blade immersed in the inner tank was rotated by a motor from above. A magnetic stirrer was placed in the bottom of the outer tank and rotated by Starcy. The stirring blade rotation speed was set to 1100 rpm, and the magnetic stirrer rotation speed was set to 250 rpm.

Using the same medium as in Example 1, the inner tank medium volume was 0.5Q,
A medium was added so that the outer tank medium volume was 0.23Q. The effective area of the partition wall at this time is 220 cm2. JTC-1 strain cells were inoculated into the inner tank. A culture medium was added to the inner tank using a peristaltic pump, and the culture medium was extracted from the outer tank so that the liquid level remained constant. The flow rate of the medium added to the inner tank is
Expressed as medium flow rate through septum per unit area: 4.7-12
．． 8mQ/cm"day.

The culture temperature is 37°C. Dissolved oxygen concentration and pH were controlled by spraying on the liquid surface using a sparger, and the flow rates of air, oxygen, and carbon dioxide were controlled so that the dissolved oxygen concentration was 2 ppm and the pH was 7.2.

When the culture was continued until the 28th day after the start of the culture, some clogging of the septum occurred. At this time, the maximum medium flow rate at which the inner tank liquid level did not change, that is, the maximum medium flow rate that could pass through the partition wall, was 5.1 mQ/cm2·day. Here, 100 mQ of culture medium was rapidly added to the outer tank, and backwashing was performed for 4 minutes at a flow rate of 160 mQ/cm2·day due to the difference in hydrostatic pressure between the liquid levels of the inner and outer tank parts. Then, the maximum allowable medium flow rate is 6.1. The flow rate recovered to mQ/cm2·day, and this flow rate was maintained for 3 days. Again, on the 31st day after the start of culture, 180 mQ of medium was rapidly added to the outer tank, and 170 mQ of medium was added to the outer tank.
When backwashing was performed for 7 minutes at a flow rate of /am2・day, the maximum flow rate of the medium that could pass was 7.0 mL/cm.
The patient recovered by 2 days. When this backwashing operation was performed every 3 days, culture could be continued for an additional month.

From the above, when using the culture tank of the present invention, even if the flow rate of the medium that can pass through the partition wall decreases due to clogging, it is possible to recover from the clogging by performing a simple backwashing operation. It was found that the recovered state could be maintained for several days.

Example 4 Using a twin-tube tapping flask, the diffusion coefficients of glucose and lactic acid in a 5US316L woven fabric material and a semipermeable membrane were determined by measurement.

A septum with an effective area of 2.1 cm, made of the above-mentioned 5US316L woven fabric material, was installed in a twin-tube tapping flask.The first tank contained DM-160AU, which does not contain glucose.
Add 0.5 g of lactic acid to the medium (10% newborn calf serum, 0.3 g IQ glutamine and Q, supplemented with 1 g/Q kanamycin).
DM-160 is added to the second tank to adjust the IQ.
80 mQ each of AU medium (10% newborn calf serum, supplemented with 0.3 g/12 glutamine and 0.1 g/fl kanamycin) was added. DM-160AU medium is prepared to have glucose of 1.0 g/[. The stirrer rotation speed was set at 40 Or p m, and the temperature was set at 37°C. Glucose and lactic acid concentrations in the first tank and second tank were measured every 2 hours.

Next, in a twin-tube tapping flask,
00, effective area consisting of a semipermeable membrane with a thickness of 200 μm1.
A 5 cm2 partition wall was installed. The first tank contained glucose-free DM-160AU medium (10% newborn calf serum, 0
．． A solution containing 3g/Q glutamine and 0.1g/Il kanamycin) with lactic acid added to 0.5g/Ω was added to the second tank, and DM-7160AU medium (10% newborn calf serum, 0.3g/Ω) was added to the second tank. glutamine and 0.1 g/fl kanamycin addition) were added at 80 mQ each. The stirrer rotation speed was set at 40 rpm, and the temperature was set at 37°C. Glucose and lactic acid concentrations in the first tank and second tank were measured every 6 hours.

The diffusion coefficients of glucose and lactic acid in the 5US316L woven fabric material and the semipermeable membrane were determined from the changes over time in the glucose and lactic acid concentrations in the first tank and the second tank. Second result
Shown in the table.

=31- Table 2 From Table 2, the diffusion coefficients of glucose and lactic acid in the 5US316L woven fabric material are 11 times and 7 times higher than those in the semipermeable membrane, respectively.
It turned out to be twice as big.

Example 5 Using the apparatus shown in FIG. 1, cells were cultured while adding a medium to the outer tank portion (corresponding to the second region) from the medium supply pipe 106.

The above 5US316L having a bottom closed cylindrical wall with a total volume of 3.0ρ in a glass culture tank with a total volume of 8.0Q.
A partition made of a woven cloth-like material was provided as shown in FIG.

The partition flange is provided with four pressure equalizing ports each having a diameter of 1.5 mmΦ. This pressure equalization port maintains the pressure difference between the inner tank and the outer tank at substantially zero.

A modified stirring blade immersed in the inner tank was rotated by a motor from above. A magnetic stirrer was placed in the bottom of the outer tank and rotated by Starcy. The stirring blade rotation speed was set to 1100 rpm, and the magnetic stirrer rotation speed was set to 180 rpm.

Using the same medium as in Example 1, the inner tank medium volume was 2.5 Il.
A culture medium was added to the outer tank so that the volume of the culture medium was 2.7Ω. The effective area of the septum at this time is 800cm''.When JTC-1 strain cells were inoculated into the inner tank, 4 x 105cm.
It became ells/m n. A culture medium was added to the outer tank using a peristaltic pump, and the culture medium was extracted from the outer tank while keeping the liquid level constant. The flow rate of the medium added to the outer tank was 1.2 to 6.712/day.

The culture temperature is 37°C. Dissolved oxygen concentration and pH
The flow rate of air, oxygen and carbon dioxide was controlled by spraying the liquid surface with a sparger so that the dissolved oxygen concentration was 2 ppm and the pH was 7.2.

Figure 6 shows the change in cell concentration in the inner tank over time. The cell concentration in the inner tank was 5.2X:]-0″cell on the 5th day after the start of culture.
s/m Q was reached, and the cell survival rate at that time was 96%. Furthermore, no outflow of cells to the outer tank was observed during the entire culture period.

From this, when using the culture tank of the present invention, it is easy to grow 5X 10
It was confirmed that high-density mass culture of cells at the 'cells/m f2 level can be achieved.

Comparative Example 1 For comparison with Example 5, a semipermeable membrane was used as the partition wall, and culture was performed using the same apparatus, method, medium, and seed cells as in Example 5.

Total capacity8. Total volume 3, Cl in a glass culture tank of OQ
A partition wall consisting of a semipermeable membrane with a molecular weight cutoff of 30,000 and a thickness of 200 μm, supported by a closed cylindrical frame at the bottom and a wire mesh, was provided as shown in FIG. The partition flange has pressure equalizing ports with a diameter of 15 mm on four sides. This pressure equalization port maintains the pressure difference between the inner tank and the outer tank at substantially zero.

Using the same medium as in Example 1, the inner tank medium volume was 2.5Ω,
A medium was added so that the outer tank medium volume was 2.7Q. The effective area of the partition wall at this time is 800 cm2. When JTC-1 strain cells were inoculated into the inner tank, 4 x 1. O'
cells/m Q. A culture medium was added to the outer tank using a peristaltic pump, and the culture medium was drawn out from the outer tank while keeping the liquid level at a constant level. The flow rate of the medium added to the outer tank is 1.2 to 6.7 ρ/day.

The culture temperature is 37°C. Dissolved oxygen concentration and pH were controlled by spraying on the liquid surface using a sparger, and the flow rates of air, oxygen, and carbon dioxide were controlled so that the dissolved oxygen concentration was 2 ppm and the pH was 7.2.

Figure 7 shows the change in cell concentration in the inner tank over time. The cell concentration in the inner tank was 2.1 at the 40th day after the start of culture. X106cells
/m Q was reached, but no further growth occurred.

Further, no outflow of cells to the outer tank was observed during the entire culture period.

From this, it was confirmed that high-density, large-scale culture of cells is difficult when a semipermeable membrane partition wall is used.

〔Effect of the invention〕

According to the present invention, it is possible to stably and continuously supply a nutrient source and remove waste products over a long period of time without damaging the cells, thereby enabling high-density, large-scale culture of cells.

[Brief explanation of the drawing]

Fig. 1 shows an example of a culture tank having a cylindrical partition with a closed bottom, Fig. 2 shows an example of a culture system, and Fig. 3 shows an example of a culture tank with a tubular spiral partition. FIG. 4 is a diagram showing an example of a culture tank having a rectangular outer tank section surrounded by plate-shaped partition walls, and FIG. 5 is a diagram showing changes in cell concentration over time in Example 2. 6 is a diagram showing the change in cell concentration over time in Example 5, and FIG. 7 is a diagram showing the change in cell concentration over time in Comparative Example 1. 101...1 culture tank, 102...inner tank part, 1
03 ... Outer tank part, 1.04 ... Partition wall, 10
5...jacket, 106...culture medium supply pipe to the outer tank section, 107...extraction pipe, 108...pump that supplies liquid to the inner tank section 102 and/or the outer tank section 103, 109. Outer tank a pump for extracting liquid from part 103;
110...Magnetic stirrer, 111...Motor 112
... Stirring shaft, 113 ... Stirring blade, 114 ...
Ring sparger 115・Nozzle, 116...Air supply pipe, 117・
...Exhaust pipe, 118 ...Bacterial filter, 11-9
...Bulkhead flange, 120 ...Pressure equalization port, 121 ・
... Level sensor, 122 ... Dissolved oxygen concentration sensor, 123 ... pH sensor, 1-24 ... Culture medium supply pipe to the inner tank section 201 - - Supply liquid storage tank, 202 - Recovery liquid storage tank, 203... Constant temperature water tank, 204... Pump that sends hot water from the constant temperature water tank 203 to the jacket 105, 205... Air mass? Low controller, 206 ... Oxygen mass flow controller, 207 ... Carbon dioxide mass flow controller, 208 ... Control computer, 20
9... Keyboard, 301... Culture tank section, 302... Inside the tube, 303
...Partition wall, 401 ...Culture tank part, 402 - Outer tank part, 403
...Bulkhead.

Claims (1)

[Scope of Claims] 1. A partition wall having pores with a diameter that allows cells to pass through allows a first area in which a culture medium in which cells are suspended to exist and a second area in which a physiological buffer or a liquid containing a nutrient source exists to be separated. cells that separate the cells, move at least one of nutrients, waste products, or products between the first region and the second region via the septum, and further move from the first region to the second region via the septum. The number of
A cell culture method characterized by suppressing liquid flow through a septum so as not to exceed the number of cells that increase in a region due to proliferation. 2. The method for culturing cells according to claim 1, characterized in that the partition wall is made of a woven material or a porous material. 3. Adding a medium to the first region and/or the second region as a method for moving at least one of nutrients, waste products, or products between the first region and the second region via the partition wall, The method for culturing cells according to claim 1, characterized in that the culture medium is extracted from the second region. 4. When the culture medium is added to the first region and the culture medium is extracted from the second region, the number of cells migrating from the first region to the second region through the partition increases due to proliferation in the first region. Claim 1, characterized in that the method for preventing the number of cells from exceeding the number of cells to be added includes controlling the flow rate of the added medium.
Method for culturing the described cells. 5. In the case where the culture medium is added to the first region and the culture medium is extracted from the second region, the second region is intermittently added to the second region through the partition wall.
The method for culturing cells according to claim 1, characterized in that a liquid flow is created from the region to the first region. 6. At least one of a nutrient source, a waste product, or a product is removed between the first region and the second region via the partition wall when the culture medium is added to the second region and the culture medium is extracted from the second region. 2. The method for culturing cells according to claim 1, wherein the method for controlling the amount of transferred medium includes controlling the flow rate of the added medium. 7. The method for culturing cells according to claim 1, wherein the method for suppressing the liquid flow through the partition wall is to reduce the pressure difference between the first region and the second region through the partition wall to substantially zero. 8. As a method for preventing the number of cells migrating from the first region to the second region via the partition wall from exceeding the number of cells increasing due to proliferation in the first region, cells floating in the first region Claim 1, characterized in that the partition wall is made of a material having an electrostatic charge of the same polarity as the electrostatic charge possessed by the
Method for culturing cells described in Section 1. 9. As a method for preventing the number of cells migrating from the first region to the second region via the septum from exceeding the number of cells increasing due to proliferation in the first region, the septum may be suspended in the first region. 2. The method for culturing cells according to claim 1, wherein a potential having the same polarity as an electrostatic charge possessed by the cells is applied. 10. A culture tank in which a first region in which a culture solution with suspended cells exists and a second region in which a solution containing a physiological buffer or a nutrient source exists are separated by a partition wall having pores with a diameter that allows the cells to pass through. means for moving at least one of a nutrient source, a waste product, or a product between the first region and the second region via the partition; A cell culturing device comprising means for suppressing liquid flow through a partition so that the number of cells does not exceed the number of cells that increases due to proliferation in the first region. 11. The cell culturing device according to claim 10, wherein the partition wall is made of a woven material or a porous material. 12. Adding a medium to the first region and/or the second region as a means for transferring at least one of nutrients, waste products, or products between the first region and the second region via the partition; 11. The cell culturing device according to claim 10, further comprising means for extracting the culture medium from the second region. 13. When the culture medium is added to the first region and the culture medium is extracted from the second region, the number of cells migrating from the first region to the second region via the partition increases due to proliferation in the first region. 11. The cell culturing apparatus according to claim 10, further comprising means for controlling the flow rate of the added medium as a means for preventing the number of cells from exceeding the number of cells. 14. In the case where the culture medium is added to the first region and the culture medium is extracted from the second region, the method is characterized by having means for intermittently creating a liquid flow from the second region to the first region via the partition wall. The cell device according to claim 10. 15. When removing the medium from the second area while adding the medium to the second area, at least one of a nutrient source, a waste product, or a product is transferred between the first area and the second area via the partition wall. 11. The cell culturing device according to claim 10, further comprising means for controlling the flow rate of the added medium as the means for controlling the amount of transferred medium. 16. The cell according to claim 10, characterized in that the means for suppressing the liquid flow through the partition wall includes means for substantially zeroing the pressure difference between the first region and the second region through the partition wall. Culture device. 17. Cells floating in the first region as a means to prevent the number of cells migrating from the first region to the second region via the partition wall from exceeding the number of cells increasing in the first region due to proliferation. 11. The cell culturing device according to claim 10, wherein the partition wall is made of a material having an electrostatic charge of the same polarity as that of the cell culture device. 18. As a means to prevent the number of cells migrating from the first region to the second region via the septum from exceeding the number of cells increasing due to proliferation in the first region, the septum is suspended in the first region. 11. The cell culturing device according to claim 10, wherein a potential having the same polarity as an electrostatic charge possessed by the cell is applied.