JPH06105680A - Method and apparatus for culturing animal cells - Google Patents

Abstract

(57) [Summary] [Structure] The culture device 1 comprises a culture tank 2, a stirring device 4 for suspending cells in a medium 10, an aeration tube 3 for supplying oxygen to the cells, and a medium 10 and cells separated from each other. The centrifuge 11 and the culture medium storage tank 14 for supplying a new culture medium to the culture tank 2 are provided. The stirring device 4 has a plurality of stirring blades 4b on the rotating shaft 4a, and these stirring blades 4b are sized so that the radius of rotation is 1/4 or more and 3/8 or less of the inner diameter of the culture tank 2. It is formed and is rotated at a rotational speed of 5 rpm or more and 30 rpm or less. [Effect] The cells are not adversely affected by damage or destruction due to physical stimulation such as shearing force due to agitation, and a large amount and high density can be cultured in the culture tank.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for culturing animal cells such as antibody-producing cells by a continuous culturing method and at the same time culturing animal cells to produce a target substance such as a monoclonal antibody. The present invention relates to a culture device.

[0002]

2. Description of the Related Art In recent years, for example, as a production means for producing biologics such as interferon, erythropoietin, colony stimulating factor, and monoclonal antibody, establishment of a method for mass-culturing animal cells on an industrial scale has become extremely important. Is coming.

Animal cells (hereinafter simply referred to as "cells")
Cultivation is carried out on a laboratory scale using culture vessels such as culture flasks and spinner flasks, but if such a small-scale culture method is simply scaled up to an industrial scale, However, it causes inconveniences such as respiratory gas exchange and agitation efficiency, and cannot mass-culture cells. Therefore, various methods for large-scale culture of cells have been conventionally studied with reference to the large-scale culture method using microorganisms.

However, cells differ from microorganisms in the following points. That is, since cells have weaker mechanical strength than microorganisms, they are easily damaged by stirring or foaming of the culture solution, require various nutrients and growth factors for growth, and have a very slow growth rate. (Doubling time
Microorganisms: 0.2 to several hours, cells: 1 to several days). Therefore, when cells are mass-cultured using a jar fermenter (stirring-type culture device) used for mass-culturing microorganisms, the cells cannot be sufficiently grown due to mechanical damage, etc. The cell density cannot be increased because the growth stops when the nutrients in the culture container are consumed. Therefore, when cells are mass-cultured using the method for mass-culturing microorganisms, there is a problem that the production efficiency of the target product is very poor.

Therefore, in order to solve these problems,
A continuous culture method called a high density perfusion culture method has been devised. The high-density perfusion culture method is to extract a supernatant (supernatant) of a cell culture solution (hereinafter, referred to as a medium) containing a target substance and a waste product to the outside of the culture tank, and a new medium containing nutrients to the culture tank. This is a method for producing the desired product by supplying the cells to the culture tank under optimal conditions for growing the cells and producing the desired product, and culturing the cells in a large amount and at high density.

As the above-mentioned conventional high-density perfusion culture method, for example, cells are enclosed in a hollow fiber cartridge having extremely fine pores, and nutrients or the like are exchanged through the hollow fibers to grow the cells. Method of producing target product
No. 2-171669), a method of immobilizing cells on the surface of a carrier made of ceramic or the like, and then proliferating the cells by exchanging nutrients or the like on the surface of the carrier to produce a desired product (Opticel (Charles River). , USA)) and the like and a jar fermenter as described above and a separation device for separating cells and medium from each other, thereby proliferating cells at a high density to produce a target product. Although a suspension culture method such as JP-A-62-134086 is proposed, the suspension culture method is easy because it is easy to sample cells, control the quality of a target substance, or increase the size of the device. Promising.

[0007]

However, in the suspension culture method, in order to keep the culture tank under optimum conditions for cell growth and production of the target substance, physical stimulation (such as shearing force by stirring the medium or Removal of (foaming etc.) has become an important issue.

[0008] The stirring of the medium is performed, for example, by rotating a stirring blade having a rotation radius of 1/4 or less of the inner diameter of the culture tank at a rotation speed of 30 rpm or more. However, there is a problem that turbulent flow is generated in the medium to give physical stimuli such as shearing force to the cells and adversely affect the cells such as damage and destruction.

As described above, in the above-mentioned conventional suspension culture method, the physical stimulation (shearing force, foaming, etc. due to stirring of the medium) to the cells is not sufficiently removed. Therefore, large-scale culture of cells has not been reached.

[0010]

Means for Solving the Problems The inventors of the present application have conducted intensive studies to solve the above problems, and as a result, completed the present invention.

That is, the method for culturing animal cells according to the invention of claim 1 is a method for culturing animal cells, which comprises culturing in a cell culture medium filled in a culture tank in order to solve the above problems. The radius of gyration is 1/4 or more of the inner diameter of the above culture tank, 3 /
The aforesaid cells are agitated with an agitator having a size of 8 or less so that the area of one agitator is 1/15 or more of the longitudinal cross-sectional area of the cell culture solution part of the culture tank. The feature is that the culture solution is stirred.

In order to solve the above-mentioned problems, the method for culturing animal cells according to a second aspect of the present invention is the method for culturing animal cells according to the first aspect, wherein the rotation speed is 800 cm / min at the outer peripheral portion of the stirring blade. It is characterized by the following.

In order to solve the above-mentioned problems, the method for culturing animal cells according to the third aspect of the present invention is the method for culturing animal cells according to the first or second aspect, wherein the cell culture medium is at least porous. In addition to supplying oxygen to animal cells by performing tube aeration with an oxygen supply means having a tube,
The cell culture solution and the animal cells are centrifuged by the separating means, the cell culture solution is drawn out of the culture tank, the animal cells are returned to the culture tank, and a new cell culture solution is added to the culture tank by the culture solution supplying means. It is characterized by supplying.

In order to solve the above-mentioned problems, an animal cell culturing apparatus according to a fourth aspect of the present invention is an animal cell culturing apparatus for culturing in a cell culture medium filled in a culture tank,
The turning radius is 1/4 or more and 3/8 or less of the inner diameter of the culture tank, and the area of one stirring blade is 1/15 or more of the longitudinal cross-sectional area of the cell culture solution part of the culture tank. It is characterized in that it is provided with a stirring means having a stirring blade formed in a size, and the cell culture solution is stirred by this stirring means.

In order to solve the above-mentioned problems, the animal cell cultivating device according to the fifth aspect of the present invention is the animal cell culturing device according to the fourth aspect, wherein the rotation speed is 800 cm / min at the outer peripheral portion of the stirring blade. It is characterized by the following.

An animal cell culture device according to a sixth aspect of the present invention is, in order to solve the above-mentioned problems, the animal cell culture device according to the fourth or fifth aspect, which has at least a porous tube, Oxygen supply means for supplying oxygen to animal cells by tube aeration in the cell culture medium and the cell culture medium and the animal cells are centrifuged and the cell culture medium is drawn out of the culture tank while the animal cells are cultured in the culture tank. It is characterized in that it is provided with a separating means for returning to the culture tank and a culture solution supplying means for supplying a new cell culture solution to the culture tank.

Examples of the above-mentioned culture method include a suspension culture method and the like. Examples of the suspension culture method include a batch culture method, a seed batch culture method (half-batch culture method, fed-batch culture method), and a continuous culture method. Examples of the continuous culture method include high-density perfusion culture method.

When the continuous culture method is carried out as the above-mentioned culture method, the cells and the medium are separated by gravity settling using a settling tube or settling tank.
There are methods such as separation using a filter and separation by centrifugation. When human antibody-producing cells are used as animal cells, the cell culture medium has a protein concentration of 5 m.
g / l or less, dissolved oxygen concentration in cell culture solution is 0.1 pp
m or more and 3 ppm or less, glucose concentration 0.1 g / l
As mentioned above, the serum-free culture solution in which the lactic acid concentration is adjusted to the range of 5 g / l or less is included.

The radius of gyration of the stirring blade in the above-mentioned culture device is in the range of 1/4 or more and 3/8 or less of the inner diameter of the culture tank. The area of one stirring blade is, for example, 1/15 or more of the vertical cross-sectional area of the cell culture solution portion of the culture tank, and specifically, it is 1/15 or more and 1/4 or less. There are things like The number of stirring blades may be two or more, and specifically, two or more and four or less.

Further, the stirring blade in the above-mentioned culture device is
The cell culture solution may be inclined at an angle of 0 ° to 15 °, preferably about 5 ° to 15 ° with respect to the rotation axis so that the cell culture solution can easily convect in the vertical direction. It may be eccentric so that the outer peripheral portion is located rearward of the inner side with respect to the vertical line from the rotation axis so that the liquid can easily flow in the centrifugal direction. Further, the length of the stirring blade in the longitudinal direction may be about 1/4 of the rotating diameter. Moreover,
The rotation speed of the stirring blade is 800c at the outer peripheral portion of the stirring blade.
Speeds of m / min or less are mentioned, and specifically, 300 cm
The speed is from 1 / min to 800 cm / min.

[0021]

According to the method of claim 1, the cell culture solution of animal cells is cultured in such a manner that the radius of gyration is 1/4 or more and 3/8 or less of the inner diameter of the culture tank, and the area of one stirring blade is the above-mentioned culture. Since the stirring is performed by a stirring means having a stirring blade formed to have a size of 1/15 or more of the longitudinal cross-sectional area of the cell culture solution portion of the tank, the animal cells are subjected to physical stimulation such as shearing force due to stirring. It is possible to carry out large-scale and high-density culture in the culture tank without being adversely affected by damage or destruction.

In the method according to the second aspect, since the rotation speed is set to 800 cm / min or less at the outer peripheral portion of the stirring blade,
Animal cells are not adversely affected by physical stimuli such as shearing force due to agitation such as damage and destruction, and a large amount and high density can be cultured in the culture tank.

In the method according to claim 3, oxygen is supplied to the animal cells by aerating the cell culture medium with oxygen at least having a porous tube to supply oxygen to the animal cells, and at the same time, the cell culture medium and the animal cells are separated by the separating means. Centrifuge and
While extracting the cell culture solution from the culture tank, the animal cells are returned to the culture tank, and a new cell culture solution is supplied to the culture tank by the culture solution supply means. In addition to being able to maintain it, animal cells are not adversely affected by physical stimuli such as shearing force due to agitation, such as damage or destruction, and can be cultured in a large amount and at high density in a culture tank.

In the structure of claim 4, the area of one agitating blade having a radius of gyration of 1/4 or more and 3/8 or less of the inner diameter of the culture vessel is the longitudinal direction of the cell culture solution part of the culture vessel. The cell culture solution is agitated by the agitation means having an agitation blade formed to have a size of not less than 1/15 of the directional cross-sectional area, and the agitation means applies a shear force to animal cells. It is possible to perform large-scale and high-density culture in a culture tank without adversely affecting physical damage such as damage or destruction.

In the structure according to the fifth aspect, since the rotation speed is 800 cm / min or less at the outer peripheral portion of the stirring blade,
It is possible to culture a large amount and high density in the culture tank without adversely affecting the animal cells such as damage or destruction due to physical stimulation such as shearing force due to stirring.

In the structure according to the sixth aspect of the present invention, at least a porous tube is provided, and the oxygen supply means for supplying oxygen to the animal cells by aeration of the tube in the cell culture solution, the cell culture solution and the animal cells are provided. The culture tank is provided with a separation means for centrifuging and extracting the cell culture solution from the culture tank while returning the animal cells to the culture tank, and a culture solution supply means for supplying a new cell culture solution to the culture tank. The inside of the cell can be maintained under optimal conditions for animal cells, and the animal cells will not be damaged or destroyed by physical stimulation such as shearing force due to agitation. It becomes possible to do.

[0027]

EXAMPLES An example of the culture apparatus of the present invention will be described below with reference to FIGS. 1 to 4.

As shown in FIG. 1, the culture apparatus 1 according to the present embodiment is provided with an aeration tube (oxygen supply means) 3 and a stirring device (stirring means) 4 inside a culture tank 2. A medium injection pipe (culture solution supply means) 5, a medium extraction pipe (separation means) 6, a cell return pipe (separation means) 7, and an exhaust pipe 8 are arranged. The above culture device 1
Has heat resistance enough to be heat-sterilized.

The culture tank 2 is made of, for example, hard glass or stainless steel, and has a cell culture solution (hereinafter,
10) is filled. The culture tank 2 is provided with a lid 2a, and the lid 2a has an aeration tube 3, a medium injection pipe 5, a medium extraction pipe 6, a cell return pipe 7,
And a hole for inserting the exhaust pipe 8 is provided.
A liquid level sensor, a DO electrode (dissolved oxygen concentration measuring device), and a pH electrode (none of which is shown) are attached to the culture tank 2 at predetermined positions. Furthermore, the culture tank 2
Is covered with a water tank, a jacket (not shown) or the like, and the temperature of the medium 10 in the culture tank 2 can be controlled by adjusting the water temperature in the water tank or the jacket. .

The components of the medium 10 are appropriately selected depending on the animal cells to be cultured (not shown, hereinafter simply referred to as "cells"). For example, RPMI-1640 medium, Dulbecco's modified Eagle medium and Ham's F- 12 medium 2:
RDF-ITES medium mixed at a ratio of 1: 1 (Murakami et al., Agric. Biol. Chem., 46 (7),
1831-1937, 1982) as a basal medium, and a serum-free medium modified by adding a trace amount of components to this basal medium is used. Since this serum-free medium has the protein concentration in the medium adjusted to 5 mg / l or less, adverse effects due to protein components are reduced when the target substance is separated from the medium, and the target substance can be easily purified. There is. Further, since the amount of expensive protein used is small, the above serum-free medium is inexpensive and economical for continuous culture.

The cells cultivated in the medium 10 are optimally selected depending on the target product to be produced. For example, when the target product is a human monoclonal antibody, human-derived B cells, mouse hybridoma, human -Lymphoid cells (human antibody-producing cells) such as mouse hybridoma and recombinant cells obtained by introducing antibody genes into myeloma cells are used. The cells introduced into the medium 10 in the culture tank 2 are grown until they reach the cell density necessary for the production of the target substance,
It is designed to produce a target product.

The aeration tube 3 has innumerable pores of a size that allows the medium 10 and cells to pass but does not allow oxygen and carbon dioxide to pass. For example, a porous fluororesin tube, a porous polypropylene tube, and a porous silicon tube. It is made of a porous tube such as a tube, is formed into a spiral shape, and is immersed in the medium 10 in the culture tank 2. The length of the aeration tube 3 is determined depending on the inner and outer diameters of the aeration tube 3, the type of cells to be cultured, the culture conditions, etc., for example, the outer diameter is 3 to 10 mm, the inner diameter is 2 to 9 mm,
In the case of a porous fluororesin tube having a porosity of 50% and a maximum pore diameter of 2 μm (for example, Poreflon: manufactured by Sumitomo Electric Co., Ltd., Osaka), the length may be 0.3 m or more, preferably 1 m or more per 1 l of the medium. . Also, ventilation tube 3
50 to 1000 ml / mi of air or oxygen
n. The air transfer rate (kLa) in this case is about 2 to 5 l / hr.

The tip portion 3a of the aeration tube 3 has the culture tank 2
While being pulled out to the outside of the culture tank 2 by being inserted into the hole provided in the lid 2a of the cell, the end portion 3 of the aeration tube 3
b is opened above the liquid surface 10a of the medium 10.
Further, the tip portion 3a of the ventilation tube 3 is connected to a blower that blows air, an oxygen cylinder (neither is shown), or the like, whereby air or oxygen is ventilated in the ventilation tube 3. . Therefore, carbon dioxide in the medium 10 and oxygen in the aeration tube 3 can be gas-exchanged by tube aeration on the surface of the aeration tube 3, and upper surface aeration can be performed on the liquid surface 10a of the medium 10. . The air or oxygen released from the end portion 3b of the aeration tube 3 is released to the outside of the culture tank 2 by the exhaust pipe 8 inserted into the hole of the lid 2a of the culture tank 2. Therefore, the aeration tube 3 can supplement the oxygen consumption in the medium 10 due to the respiration of the cells to promote the growth of the cells and produce the target product.

Further, since the cell density in the medium 10 is low and the oxygen consumption is small at the start of cell culture, the oxygen supply amount may be small. Therefore, air may be ventilated in the vent tube 3 to maintain the dissolved oxygen concentration at 2 to 6 ppm. Then, in the logarithmic growth phase, the cell density becomes high and the oxygen consumption amount is large, so that it is necessary to increase the oxygen supply amount as well. Therefore, oxygen may be ventilated in the vent tube 3 to control the dissolved oxygen concentration to 0.1 to 3 ppm.

Further, the tip portion 3a of the ventilation tube 3 is
It is also connected to a carbon dioxide cylinder (not shown), and the pH of the medium 10 can be adjusted by ventilating carbon dioxide in the vent tube 3. It should be noted that the end portion 3b of the aeration tube 3 is not opened above the liquid surface 10a of the culture medium 10 and is inserted into a hole provided in the lid 2a of the culture tank 2 so as to be drawn out of the culture tank 2; A ventilation pipe (not shown) may be attached to 2a to supply air or oxygen separately from the ventilation tube 3 so that the liquid surface 10a of the culture medium 10 is vented on the upper surface.

The stirring device 4 is made of, for example, stainless steel or fluororesin, and the rotary shaft 4a is inserted into a hole provided at the center of the bottom surface of the culture tank 2 so that the culture tank 2 can be obtained.
It is pulled out to the outside and connected to a stirring motor (not shown). At least 2 at the tip of the rotary shaft 4a.
A number of stirring blades 4b ... Are attached. Although the shape of the stirring blade 4b is not particularly limited, as shown in FIG. 2, for example, when the stirring blade 4b is formed in a flat plate shape, the cells are not damaged and the cells are rotated at a rotation speed described below. In order to suspend in the medium 10, the blade length A of the stirring blade 4b (that is, the radius of gyration of the stirring blade 4b) is 1 / inner diameter C of the culture tank 2.
The length may be 4 or more and 3/8 or less.

The height B of the stirring blade 4b is the same as that of the stirring blade 4b.
Area (A × B) of the medium is 10 or more, preferably 1/15 or more and 1/4 or less of the longitudinal sectional area of the medium 10 when the culture tank 2 is cut in the longitudinal direction. The height B is about 1 / the blade length A, although it depends on the liquid depth of the medium 10 filled in the culture tank 2.
It may be formed to have a height that is 2 to 2 times. The stirring blades 4b are attached to the rotary shaft 4a at an inclination angle of 0 to 15 °, preferably 5 to 15 ° so that the medium 10 can easily convect in the vertical direction. Has become. The stirring blades 4b may be attached so as to be eccentric so that the outer peripheral portion is located rearward from the inner side with respect to the vertical line from the rotating shaft 4a so that the culture medium 10 can easily flow in the centrifugal direction. Further, the rotary shaft 4a is provided in a direction perpendicular to the bottom of the culture tank 2 and, in order to facilitate convection of the medium 10 in the vertical direction, the rotary shaft 4a is 20 ° or less from the vertical direction with respect to the bottom of the culture tank 2. May be provided at an angle.

In order to suspend the cells in the medium 10 without damaging the cells, the stirring blades 4b ... Rotate at a peripheral speed of 800 cm / min.
Below, preferably, 300 cm / min. Above, 800
cm / min. The stirring blades 4b, ..., Specifically, the stirring blades 4b ... The culture medium 10 is 5 rpm or more and 30 rpm or less, preferably 10 rpm.
It is designed to stir at a rotation speed of m.m. or more and 20 rpm or less. Here, if the blade length A of the stirring blade 4b is the culture tank 2
If the length is less than 1/4 of the inner diameter C of
Even if the stirring blades 4b ... Are rotated at a rotation speed of 30 rpm, the cells cannot be suspended in the medium 10, which is not preferable, and the blade length A of the stirring blades 4b is 3 which is the inner diameter C of the culture tank 2. If it is formed to have a length larger than / 8, turbulent flow occurs in the medium 10 between the blade tip of the stirring blade 4b and the inner wall of the culture vessel 2 and damages the cells, which is not preferable. Therefore, the cells are suspended in the medium 10 without damaging the cells by setting the blade length A of the stirring blades 4b as described above and controlling the rotation speed of the stirring blades 4b. You can do it.

The rotating shaft 4a of the stirring device 4 is the culture tank 2
Instead of being pulled out to the outside of the culture tank 2 by being inserted into the hole provided in the bottom surface, it may be pulled out to the outside of the culture tank 2 by being inserted into the hole provided in the lid 2 a of the culture tank 2. In this case, since it is not necessary to provide a hole on the bottom surface of the culture tank 2, the structure of the culture tank 2 can be simplified.

As shown in FIG. 1, the medium extracting tube 6 and the cell returning tube 7 are made of, for example, stainless steel or fluororesin, and are inserted into the holes provided in the lid 2a of the culture tank 2. By doing so, it is drawn out of the culture tank 2. These pipes 6 and 7 are connected to a centrifuge (separation means) 11 via a pump or the like (not shown) to separate the cells suspended in the medium 10 and the target substance produced by the cells. After separation, the medium is exchanged and the target product is collected. That is, the supernatant (supernatant) of the medium 10 is extracted through the medium extracting pipe 6, the cells and the target substance are centrifuged by the centrifuge 11, and the medium containing the cells is transferred through the cell returning pipe 7 to the bottom of the culture tank 2. By returning the medium to the vicinity and feeding the medium containing the target substance to the target substance storage tank (separation means) 13 through the liquid feeding pipe 12, the medium is replaced and at the same time the target substance is recovered. The target substance contained in the medium stored in the target substance storage tank 13 is isolated and purified by a predetermined method.

The centrifuge 11 described above separates the supernatant of the medium 10 from 300 xg or less, preferably 100 xg, in order to separate the cells from the target substance without damaging the cells.
It is designed to perform centrifugal separation at the following centrifugal acceleration.
The supernatant of the medium 10 may be extracted continuously or intermittently. For example, when the culture is in a steady state and the supernatant of the medium 10 is continuously extracted, depending on the capacity of the culture tank 2 and the type of cells to be cultured, the centrifuge 11 may have a volume of 1 to 20 l / l. It suffices that it has the ability to process the liquid volume of h under aseptic conditions (for example, Centritech CC100: Centritech AB, Northborg, Sweden).

The above-mentioned culture medium injection pipe 5 is connected to a culture medium storage tank (culture liquid supply means) 14 containing a new culture medium, and the inside of the culture tank 2 is opened through a hole provided in the lid 2a of the culture tank 2. Has been inserted into. Then, the medium injection pipe 5 is configured to supply the culture tank 2 with a new medium in the same amount as the medium containing the target substance sent to the target substance liquid storage tank 13. The supply of new medium may be performed continuously or intermittently. However, if the culture reaches a steady state, it is desirable to carry out the culture continuously.

The liquid level sensor (not shown) detects the position of the liquid level 10a of the medium 10 in the culture tank 2,
The result is input to a control device (not shown). Similarly,
A pH electrode (not shown) measures the pH of the medium 10, and a DO electrode (not shown) measures the dissolved oxygen concentration in the medium 10 and inputs the results to the controller. As a result, the control device adjusts the amount of new medium supplied from the medium injection pipe 5 or the amount of medium extracted from the medium extraction pipe 6, or the amount of oxygen or carbon dioxide aerated in the aeration tube 3. Then, the dissolved oxygen concentration in the medium 10 is controlled to 0.1 ppm or more and 3 ppm or less, the glucose concentration is controlled to 0.1 g / l or more, and the lactic acid concentration is controlled to 5 g / l or less. 10 is kept in an optimal culture environment for growing cells and producing the desired product.

For example, the dissolved oxygen concentration in the medium 10 is 0.
If it is less than 1 ppm, the cells will be in an oxygen-deficient state, which is not preferable, while if the dissolved oxygen concentration is more than 3 ppm, the cells may be damaged by excess oxygen, which is not preferable. If the glucose concentration is less than 0.1 g / l, the cells will not grow sufficiently, which is not preferable. On the other hand, if the lactate concentration is more than 5 g / l, the toxicity to the cells will be increased and the cell activity will decrease, resulting in a survival rate. Is reduced, which is not preferable. The exchange rate of the medium 10 (perfusion ratio)
Depending on the type of cells to be cultivated, cell density, etc., it may be about 0.5 to 5 times / day with respect to the amount of medium in the culture tank 2.

Next, as one embodiment of the culturing method of the present invention, continuous culturing of cells is carried out by using the culturing apparatus 1 having the above-mentioned structure, and the cell density in the medium 10 and the medium of the target substance produced by the cells are obtained. The results of measuring the concentration in 10 for several days under the following conditions are shown.

The above conditions are as follows: 1) RD as the medium 10
A serum-free low protein medium obtained by modifying F-ITES medium,
The S-RDF-ITES medium having the composition as shown in Table 1 was used. Human transferrin (2.
0 mg / l) and bovine insulin (2.0 mg / l)
l) is a protein component, and therefore S-RDF-ITES
The protein concentration of the medium was adjusted to 4.0 mg / l. The glucose concentration is adjusted to 5.0 g / l.

[0047]

[Table 1]

2) As the culture tank 2 of the culture device 1,
A flat bottom jar fermenter (FZ2000: manufactured by Alfa Laval, Sweden) having an inner diameter of 250 mm, a height of 480 mm and a capacity of 20 l was used. The temperature control of the medium in the flat bottom jar fermenter was performed by adjusting the temperature of the water passed through the tube in the jacket covering the outside of the jar fermenter. 3) As the stirring blades 4b ..., four blades having a blade length A of 80 mm and a height B of 160 mm are used as the stirring blades 4b, and are attached at an inclination angle of 5 ° with respect to the rotating shaft 4a. It was rotated at a rotation speed of -15 rpm. 4) As the ventilation tube 3, 5 m of a porous fluororesin tube (Biot, Tokyo) having an outer diameter of 7 mm was spirally wound and placed in a flat-bottomed jar fermenter. 5)
A liquid level sensor (glass light sensor: manufactured by Fujiwara Seisakusho, Tokyo), a pH electrode (465-50-S7: manufactured by Ingold, Switzerland), and a DO electrode (φ25 mm-70: manufactured by Ingold, Switzerland) ) Was attached.

Furthermore, 6) culturing the cells, before carrying out the main culture in the culturing apparatus 1, a culture flask, a 1 l round bottom flask,
Preculture was performed using a 6-liter round bottom flask. That is, the cells to be main-cultured in the culture device 1 are a culture flask,
Using 1 liter and 6 liter round bottom flasks, cells were inoculated at a predetermined cell density and then grown by batch culture. The three types of flasks used for the pre-culture will be described below.

As a culture flask (not shown), a volume of 1
A 50 ml static tissue culture flask (T-flask: Corning, USA) was used.

As shown in FIG. 3, a glass spinner flask (Shibata Hario Glass, Tokyo) 22 having an inner diameter of 100 mm and a height of 200 mm was used as a 1 l round bottom flask. As the stirring blades 24b of the stirring device 24 in the spinner flask 22, the blade length A ₁ formed in a flat plate shape is 4
Two blades having a diameter of 0 mm and a height B ₁ of 20 mm were used. As the aeration method, upper surface aeration for substituting the gas in the flask was performed.
In addition, a pH electrode (405-DPAS-K8S / 325: manufactured by Ingot, not shown) is attached to the spinner flask 22.
Switzerland). In addition, the spinner flask 22
The temperature of the medium inside was controlled by adjusting the water temperature of a water tank (not shown) covering the outside of the spinner flask 22.

As shown in FIG. 4, a glass round-bottom jar fermenter (Shibata Hario Glass, Tokyo) having an inner diameter of 190 mm and a height of 250 mm was used as a 6-liter round-bottomed flask 32.
Was used. As the stirring blades 34b of the stirring device 34 in the round-bottom jar fermenter 32, four blades having a blade length A ₂ of 40 mm and a height B ₂ of 80 mm formed in a flat plate shape are used, and It was attached at an inclination angle of 5 °. As a ventilation tube 33 for performing tube ventilation, 3 m of a porous fluororesin tube (Biot, Tokyo) having an outer diameter of 7 mm was spirally wound and placed in a round bottom jar fermenter 32. In addition, the pH electrode (405-DPAS-K8) was attached to the round bottom jar fermenter 32.
S / 325: Ingold, Switzerland) 39a and D
O electrode (φ14mm: Maruhishi Bioengine, Tokyo) 39
b was attached. In addition, round bottom jar fermenter 3
The temperature control of the medium in 2 was performed by adjusting the water temperature of the water tank (not shown) covering the outside of the jar fermenter 32. The culture environment such as the stirring speed and pH of the stirring device 34 was controlled by using a control device (MCT-3S: manufactured by Maruhishi Bioengine, Tokyo) not shown.

Then, under the conditions 1) to 6) above, the cells were suspended at a predetermined cell density in the S-RDF-ITES medium (medium 10) in a flat-bottomed jar fermenter (culture tank 2), Main culture was started. In addition, as cells grow,
Culture was continued by performing medium exchange or continuous perfusion using the centrifuge 11. The results are shown below as Examples 1 to 3. However, the culture method of the present invention is not limited to Examples 1 to 3.

[Example 1] As a cell, a human Ig (immunoglobulin) M producing human-mouse hybridoma cell line obtained by cell fusion of mouse myeloma P3X63Ag8.653 and human lymphocytes was used.

First, the cells are placed in a medium 70 in a T-flask.
Inoculate the cells at a cell density of 1.0 × 10 ⁵ cells / ml and suspend the cells in an atmosphere of 5% carbon dioxide concentration.
When left standing at ℃, cell density of 7.4
Proliferated to × 10 ⁵ cells / ml. The antibody (target substance, human IgM) concentration at the end of culture was 28 mg / l.

Next, the cells grown in the T-flask were
Inoculate 500 ml of the medium in the spinner flask 22 so that the cell density becomes 1.0 × 10 ⁵ cells / ml, and suspend.
When stirred at 37 ° C., pH 6.8 to 7.1, and rotation speed of 30 rpm, the cell density was 6.6 × 10 3 days after the start of culture.
Proliferated to ⁵ cells / ml. The antibody concentration at the end of culture was 25 mg / l.

Further, the cells grown in the spinner flask 22 were mixed with 5 l of the medium in the round bottom jar fermenter 32.
Inoculate the cells to a cell density of 1.0 × 10 ⁵ cells / ml, suspend and incubate at 37 ° C., pH 6.8-7.1, 13 rpm.
When agitated at a rotation speed of, the cells grew to a cell density of 7.9 × 10 ⁵ cells / ml 3 days after the start of culture. The antibody concentration at the end of culture was 30 mg / l.

The cells precultured as described above were added to 15 liters of medium in a flat-bottomed jar fermenter at a cell density of 1.
Inoculate and suspend at 8 × 10 ⁵ cells / ml, and
The main culture was carried out at a temperature of 6.8 to 7.1 and a rotation speed of 10 rpm. Further, the aeration rate in the porous fluororesin tube is set to 200 to 500 ml / min. At the beginning of the main culture, air was ventilated in the tube to adjust the dissolved oxygen concentration to 2
It was maintained at 6 ppm, and as the cells grew, the oxygen partial pressure in the air was increased to control the dissolved oxygen concentration to 0.5 to 3 ppm. Then, the cell density of 6.
It grew to 8 × 10 ⁵ cells / ml. At this point, perfusion was performed for 4 hours, the medium was exchanged, and further perfusion was continuously performed 4 days after the start of the main culture. In addition, the exchange rate (perfusion ratio) of the medium was determined by measuring the amount of the medium solution (15) in the flat bottom jar fermenter.
1) to 1.3 times / day. Further, during the main culture period, the glucose concentration in the medium was controlled to 1 g / l or more and the lactic acid concentration was controlled to 2.7 g / l or less.

When the cells were subjected to the main culture for 15 days as described above, the cells were grown to have the cell densities shown in Table 2, and 13 days after the start of the main culture, the cell density was 7.7 × 10 ⁶ cells / ml. became. In addition, as the cells proliferated, the antibody concentration also increased as shown in Table 2, and the antibody concentration reached 105 mg / l 12 days after the start of the main culture.

[0060]

[Table 2]

From the above results, it is understood that the culturing apparatus 1 and the culturing method configured as described above promote the growth of cells and the improvement of the productivity of the target substance. That is, with the culture device 1 and the culture method having the above-mentioned configurations, it is possible to grow cells without damaging them, and at the same time, the cells are cultured in a culture tank in a large amount and at high density to obtain the target antibody. It can be seen that it is possible to mass-produce in a stable and efficient manner.

Example 2 As a cell, a mouse IgG-producing mouse hybridoma cell line obtained by cell fusion of mouse myeloma P3X63Ag8.653 and mouse lymphocytes was used.

First, the cells are placed in a medium 70 in a T-flask.
Inoculate and suspend the cells at a cell density of 1.1 × 10 ⁵ cells / ml, and incubate them in an atmosphere with a carbon dioxide concentration of 5%.
When left to stand at ℃, cell density of 1.1
Proliferated to 5 × 10 ⁶ cells / ml. Cell doubling time is 2
It was 8.1 hours.

Next, the cells grown in the T-flask were
500 ml of the medium in the spinner flask 22 was inoculated and suspended at a cell density of 2.7 × 10 ⁵ cells / ml,
When stirred at 37 ° C., pH 6.8 to 7.1, and rotation speed of 20 rpm, the cell density was 8.2 × 10 3 days after the start of culture.
Proliferated to ⁵ cells / ml. The doubling time of the cells was 44.6 hours.

Further, the cells grown in the spinner flask 22 were treated with 5 l of the medium in the round bottom jar fermenter 32.
Inoculate the cells to a cell density of 3.1 × 10 ⁵ cells / ml, suspend and incubate at 37 ° C., pH 6.8-7.1, 12 rpm.
When the cells were stirred at a rotation speed of 1, the cells grew to a cell density of 1.05 × 10 ⁶ cells / ml on the second day after the start of culture. The doubling time of the cells was 27.0 hours.

The cells precultured as described above were inoculated and suspended in 14.5 l of medium in a flat-bottomed jar fermenter at a cell density of 2.9 × 10 ⁵ cells / ml,
Main culture was carried out at 37 ° C., pH 6.8 to 7.1, with rotation speeds of 10 rpm. Further, the aeration rate in the porous fluororesin tube is set to 200 to 500 ml / min. At the beginning of the main culture, the dissolved oxygen concentration is kept at 2 to 6 ppm by aerating air in the tube, and as the cells grow, the oxygen partial pressure in the air is increased to raise the dissolved oxygen concentration to 0.5 to 3 ppm.
Controlled to. Then, 4 days after the start of the main culture, the cells grew to a cell density of 1.08 × 10 ⁶ cells / ml. At this point, perfusion was performed for 3 hours, and the medium was replaced. Also, after the start of main culture, 5
On the day, the cells grew to a cell density of 1.33 × 10 ⁶ cells / ml. At this point, perfusion was performed for 3 hours, the medium was exchanged, and further perfusion was continuously performed 6 days after the start of the main culture.
The medium exchange rate (perfusion ratio) was 1.5 to 2. with respect to the medium liquid amount (14.5 l) in the flat bottom jar fermenter.
It was set to 0 times / day. Also, during the main culture period, the glucose concentration in the medium was 1.5 g / l or more, and the lactic acid concentration was 2.3 g / l.
The following are controlled respectively.

When the cells were subjected to the main culture for 18 days as described above, the cells were grown to have the cell densities shown in Table 3. On the 18th day after the start of the main culture, the cell density was 3.4 × 10 ⁶ cells / ml. became.

[0068]

[Table 3]

From the above results, it is understood that the culturing apparatus 1 and the culturing method configured as described above promote the growth of cells. That is, the culture apparatus 1 and the culture method configured as described above allow cells to be proliferated without damaging them, and also capable of culturing a large number of cells at high density in a culture tank. Recognize.

[Example 3] As cells, human IgM-producing recombinant cells using Namalwa cells as hosts were used.

First, the cells are placed in a medium 70 in a T-flask.
Inoculate and suspend the cells at a cell density of 2.0 × 10 ⁵ cells / ml, and incubate them in an atmosphere with a carbon dioxide concentration of 5%.
When left still at 0 ° C, the cell density was 9.4 days after the start of culture.
Proliferated to × 10 ⁵ cells / ml. The cell doubling time is 43.
It was 7 hours. At the end of the culture, the antibody (target
The human IgM) concentration was 5.8 mg / l.

Next, the cells grown in the T-flask were
Inoculate 500 ml of the medium in the spinner flask 22 to a cell density of 2.1 × 10 ⁵ cells / ml and suspend,
When stirred at 37 ° C., pH 6.8 to 7.1, and rotation speed of 15 rpm, the cell density was 1.27 × 1 on the 4th day after the start of culture.
0 were grown in ^six / ml. The doubling time of the cells was 33.8 hours. The antibody concentration at the end of culture is 3 mg /
It was l.

Further, the cells grown in the spinner flask 22 were treated with 5 l of the medium in the round bottom jar fermenter 32.
To a cell density of 2.6 × 10 ⁵ cells / ml and suspended, and 37 ° C., pH 6.8-7.1, 15 rpm.
When the cells were stirred at a rotation speed of, the cells grew to a cell density of 7.9 × 10 ⁵ cells / ml 5 days after the start of culture. The doubling time of cells was 71.6 hours. The antibody concentration at the end of culture was 3.9 mg / l.

The cells precultured as described above were inoculated and suspended in 15.8 l of a medium in a flat-bottomed jar fermenter at a cell density of 2.3 × 10 ⁵ cells / ml,
Main culture was carried out at 37 ° C., pH 6.8 to 7.1, with rotation speeds of 10 rpm. Further, the aeration rate in the porous fluororesin tube is set to 200 to 500 ml / min. At the beginning of the main culture, the dissolved oxygen concentration is kept at 2 to 6 ppm by aerating air in the tube, and as the cells grow, the oxygen partial pressure in the air is increased to raise the dissolved oxygen concentration to 0.5 to 3 ppm.
Controlled to. Then, 3 days after the start of the main culture, the cells grew to a cell density of 9.2 × 10 ⁵ cells / ml. At this point, perfusion was performed for 3 hours, and the medium was replaced. 5 days after the start of main culture,
The cells were grown to a cell density of 1.7 × 10 ⁶ cells / ml. At this point, perfusion was performed for 3 hours, and the medium was replaced. 6 after the start of main culture
On the day, the cells were grown to a cell density of 2.7 × 10 ⁶ cells / ml.
At this point, perfusion was performed for 3 hours, and the medium was replaced. Seven days after the start of the main culture, the cells grew to a cell density of 4.1 × 10 ⁶ cells / ml. At this point, perfusion was performed for 3 hours, the medium was replaced,
Furthermore, continuous perfusion was performed 8 days after the start of the main culture. The exchange rate (perfusion ratio) of the medium was 1.5 to the amount of medium liquid (15.8 l) in the flat bottom jar fermenter.
5 times / day. During the main culture period, the glucose concentration in the medium was 1.5 g / l or more, and the lactic acid concentration was 2.5 g / l.
The following are controlled respectively.

When the cells were subjected to the main culture for 21 days as described above, they were grown to the cell densities shown in Table 4, and 20 days after the start of the main culture, the cell density was 1.8 × 10 ⁷ cells / ml. became. Further, as the cells grew, the antibody concentration also increased as shown in Table 4, and the antibody concentration on the 11th day after the start of the main culture was stable at 10 to 19 mg / l.

[0076]

[Table 4]

From the above results, it is understood that the culturing apparatus 1 and the culturing method configured as described above promote the growth of cells and the improvement of the productivity of the target substance. That is, with the culture device 1 and the culture method having the above-mentioned configurations, it is possible to grow cells without damaging them, and at the same time, the cells are cultured in a culture tank in a large amount and at high density to obtain the target antibody. It can be seen that it is possible to mass-produce in a stable and efficient manner.

The above-mentioned Examples 1 to 3 show an example of cell culture using the culture method and the culture apparatus of the present invention. The dimensions of the culture tank 2, the preculture method and apparatus,
Of course, the composition of the medium 10, the type of cells, the culture environment such as the temperature and pH of the medium, and the like, are not limited to the above Examples 1 to 3.
It is not limited to the numerical values, types, etc. shown in, and can be appropriately changed as necessary. In addition, the number of days (period) of culturing the cells is not, of course, one that is terminated or discontinued in the number of days shown in Examples 1 to 3 above, and the cells can be cultured for several months or using the culture method and the culture device of the present invention. It is possible to continuously culture for several years.

[0079]

As described above, in the method for culturing animal cells according to the present invention, as described above, the radius of gyration is 1 of the inner diameter of the culture tank.
/ 4 or more and 3/8 or less, having an agitating blade formed to have a size such that the area of one agitating blade is 1/15 or more of the vertical cross-sectional area of the cell culture solution part of the above-mentioned culture tank This is a method of stirring the above-mentioned cell culture solution with a stirring means.

As a result, it is possible to cultivate a large amount of animal cells in a culture tank at a high density without being adversely affected by damage or destruction due to physical stimulation such as shearing force caused by stirring. Play.

As described above, the method for culturing animal cells according to the second aspect of the present invention is the method for culturing animal cells according to the first aspect, wherein the rotation speed is 800 cm / cm at the outer peripheral portion of the stirring blade.
This is a method of setting it to less than a minute.

As a result, the animal cells are not adversely affected by damage or destruction due to physical stimulation such as shearing force due to agitation, and it is possible to culture a large amount and high density in the culture tank. Play.

As described above, the method for culturing animal cells according to the invention of claim 3 is the method for culturing animal cells according to claim 1 or 2, wherein oxygen having at least a porous tube in the cell culture medium is used. The supply means vents the tube to supply oxygen to the animal cells, and the separating means centrifuges the cell culture solution and the animal cells to draw the cell culture solution out of the culture tank while returning the animal cells to the culture tank. And
This is a method of supplying a new cell culture solution to the culture tank by the culture solution supply means.

As a result, the inside of the culture tank can be maintained under optimal conditions for animal cells, and the animal cells are not adversely affected by physical stimulation such as shearing force due to agitation such as damage or destruction. There is an effect that it becomes possible to culture a large amount and a high density inside.

In the apparatus for culturing animal cells according to the fourth aspect of the invention, as described above, the radius of gyration is 1 / the inner diameter of the culture tank.
Agitation with 4 or more and 3/8 or less agitating blades formed in such a size that the area of one agitating blade is 1/15 or more of the longitudinal cross-sectional area of the cell culture solution part of the above-mentioned culture tank A means is provided, and the cell culture solution is agitated by the agitating means.

As a result, it is possible to cultivate animal cells in a large amount and at high density in a culture tank without adversely affecting physical damage such as shearing force caused by agitation or physical damage such as destruction. Play.

As described above, the apparatus for culturing animal cells according to the fifth aspect of the invention is the apparatus for culturing animal cells according to the fourth aspect, wherein the rotation speed is 800 cm / in the outer peripheral portion of the stirring blade.
The configuration is less than a minute.

As a result, it becomes possible to culture a large amount and high density in the culture tank without adversely affecting animal cells such as damage or destruction due to physical stimulation such as shearing force due to stirring. Play.

As described above, the device for culturing animal cells according to the invention of claim 6 is the device for culturing animal cells according to claim 4 or 5, which has at least a porous tube and is in a cell culture medium. Oxygen supply means for supplying oxygen to animal cells by tube aeration and centrifugation of the cell culture solution and the animal cells to extract the cell culture solution from the culture tank while returning the animal cells to the culture tank And a culture solution supply means for supplying a new cell culture solution to the culture tank.

As a result, the inside of the culture tank can be maintained under optimal conditions for animal cells, and the animal cells are not adversely affected by physical stimulation such as shearing force due to agitation such as damage or destruction. There is an effect that it becomes possible to culture a large amount and a high density inside.

[Brief description of drawings]

FIG. 1 is a block diagram showing a configuration of a culture device according to an embodiment of the present invention.

FIG. 2 is an explanatory view showing the size of a stirring blade in the culture device.

FIG. 3 is an explanatory diagram of a spinner flask used for preculture.

FIG. 4 is an explanatory diagram of a round bottom jar fermenter used for preculture.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Culture device 2 Culture tank 3 Aeration tube (oxygen supply means) 4 Stirring device (stirring means) 4b Stirring blade 5 Medium injection pipe (culture liquid supply means) 6 Medium extraction pipe (separation means) 7 Cell return pipe (separation means) 10 medium (cell culture medium) 11 centrifuge (separation means) 13 target liquid storage tank (separation means) 14 medium storage tank (culture liquid supply means) A blade length (that is, radius of gyration) B height C inner diameter

(72) Inventor Hironori Ishikawa 4-2-1 Takashi, Takarazuka-shi, Hyogo Sumitomo Chemical Co., Ltd. (72) Inventor Hiroshi Noguchi 4-2-1 Takashi, Takarazuka-shi, Hyogo Industry Co., Ltd.

Claims (6)

[Claims] 1. A method for culturing animal cells, which is cultivated in a cell culture medium filled in a culture tank, wherein a radius of gyration is 1/4 or more and 3/8 or less of an inner diameter of the culture tank, and a stirring blade 1 It is possible to stir the above-mentioned cell culture liquid with a stirring means having a stirring blade formed to have a size such that the area of one plate is 1/15 or more of the longitudinal sectional area of the cell culture liquid part of the above-mentioned culture tank. Characterized method of culturing animal cells. 2. The rotation speed is 800 cm at the outer peripheral portion of the stirring blade.
2. The method for culturing animal cells according to claim 1, wherein the rate is not more than 1 / min. 3. A cell culture solution is provided with at least a porous tube to aerate the cells with oxygen to supply oxygen to the animal cells, and the separation means separates the cell culture solution from the animal cells by centrifugation to separate the cells. 3. The method of claim 1 or 2, wherein the culture solution is drawn out of the culture tank, the animal cells are returned to the culture tank, and a new cell culture solution is supplied to the culture tank by the culture solution supply means. Method for culturing animal cells. 4. An apparatus for culturing animal cells, which is cultivated in a cell culture medium filled in a culture tank, wherein a turning radius is 1/4 or more and 3/8 or less of the inner diameter of the culture tank. The cell culture device is provided with a stirring means having a stirring blade formed to have a size such that the area of the sheet is 1/15 or more of the longitudinal cross-sectional area of the cell culture solution part of the culture tank. An apparatus for culturing animal cells, which comprises stirring a liquid. 5. The rotation speed is 800 cm at the outer peripheral portion of the stirring blade.
5. The animal cell culture device according to claim 4, wherein the culture rate is not more than 1 minute. 6. A cell culture medium which has at least a porous tube and which is provided with oxygen supply means for supplying oxygen to animal cells by aerating the cell culture medium with a tube and centrifuging the cell culture medium and the animal cell. 5. The method according to claim 4, further comprising a separating means for returning the animal cells to the culture tank and a culture solution supplying means for supplying a new cell culture solution to the culture tank while extracting the cells from the culture tank. 5. The animal cell culture device according to 5.