JPH0640831B2 - Method for producing culture medium containing physiologically active substance secreted from cells - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a culture solution containing a physiologically active substance secreted from cells.

[Conventional technology]

The physiologically active substance is a substance that gives a certain activity to a cell or tissue of a living body to cause a specific physiological reaction in the cell or tissue, and some may be artificially synthesized.
Normally, one has to wait for the production of living cells to maintain them alive.

As a conventional method for producing a physiologically active substance, a method of culturing cells that produce a target physiologically active substance in order to keep them alive is used.

As a method for culturing cells, for example, a method of culturing in the body of an animal such as pseudomouse (Japanese Patent Laid-Open No. 54-98307) and a method of culturing using a medium are known. The former method has a problem that the amount of the physiologically active substance obtained from one animal is extremely small and the efficiency is very low. On the other hand, in the latter culture method using a medium, when the producing cells that produce the physiologically active substance are suspension-proliferating cells, that is, cells that are capable of sustaining survival in the state where the cells themselves are suspended in a liquid medium, , Can be brought into contact with the medium by suspending it in a liquid medium which is a nutrient source, or when the cells are adhesion-dependent cells, that is, when adhesion to a substrate is essential for survival and maintenance in the liquid medium, It is necessary to attach the cells to the surface of a suitable substrate and then contact them with liquid medium.
As a substrate for adhering adhesion-dependent cells, a large adhesion area can be easily obtained, and thus, carrier particles having a small diameter have recently been used. As a method for culturing cells as described above, a method of culturing the liquid medium while stirring the liquid medium by a mechanical stirring mechanism such as a rotary blade (Japanese Patent Laid-Open No.
-100223), a method for culturing cells on a microcarrier while stirring a liquid medium in the same manner (JP-A-59-1465).
No. 98), and others are known.

[Problems to be solved by the invention]

In order to obtain a physiologically active substance using the culture method using a medium as described above, it is necessary to keep the producing cells alive, and under the condition that the physiologically active substance is produced with high efficiency, It is essential to create an environment in which the cells are constantly in contact with fresh medium. For that purpose, it is necessary to disperse the producing cells in a liquid medium with a high degree of dispersion.

Also, when the producing cells are adhesion-dependent cells, when the adhesion-dependent cells are adhered to the carrier particles as a substrate,
If the carrier particles can be dispersed at a high degree of dispersion in the liquid medium to which the cells are added, the surface of the carrier particles will be effectively used on average, and eventually the adhesion will be high with a high adhesion rate. Can be achieved, which is extremely advantageous in increasing the production efficiency of the physiologically active substance.

Conventionally, as a method for constantly contacting cells with a new liquid medium, as in cell culture, a method in which a system in which cells are added to a liquid medium is stirred by a rotary blade is generally used. Since the method is to apply shearing force to a mixed system consisting of liquid medium and cells, it is impossible to avoid damage to the cells in culture due to collision with a rotor or by the action of large shearing force. Therefore, the production efficiency of the physiologically active substance is low, and this method may not be used depending on the cell type. In addition, in order to carry out this method on a large scale, a large-capacity device is required and a large agitator is also required, and as a result, the shearing force is inevitably increased, the production of the physiologically active substance of interest The efficiency will be further reduced.

As described above, it can be said that obtaining a physiologically active substance by culturing cells is basically advantageous, but in reality, the production cells are always contacted with a new medium. Moreover, it has been difficult to obtain a living environment condition in which the physiologically active substance-producing action of the cells is exhibited with high efficiency. The present invention is intended to solve such a problem.

[Means for solving problems]

In the present invention, a liquid medium and a mixed system consisting of dispersed particles to which adhesion-dependent cells producing a physiologically active substance are adhered or suspension-proliferating cells producing a physiologically active substance are filled with a filling ratio of 98% by volume or more. By rotating the culture vessel filled so as to rotate about a substantially horizontal axis, or by rotating the culture vessel about the axis while being separated from the substantially horizontal axis. The system is rotated together with the culture vessel, thereby moving the dispersed particles or suspension proliferating cells in the mixed system, thereby dispersing the dispersed particles or suspension proliferating cells in the liquid medium. Comprising a physiologically active substance secreted from cells by a method characterized by comprising the step of maintaining the adhesion-dependent cells or suspension-proliferating cells alive. To produce a nutrient solution.

The present invention will be specifically described below.

In the present invention, as shown in FIG. 1 and FIG.
For example, a cylindrical closed container 1 is filled with a liquid medium 3 containing production cells 2 that produce a physiologically active substance so that the internal space of the container 1 is substantially filled, and the axis X of the container 1 is filled.
Is kept substantially horizontal and the container 1 is allowed to rotate about this axis X at a constant rotation speed. Then, by rotating the container 1, after the initial period immediately after the start of rotation,
The mixed system of the internal liquid medium 3 and the producing cells 2 is the liquid medium 3
By virtue of the viscosity of, the container 1 and the container 1 are made to rotate steadily around the axis X without any mechanical flow.

In this way, in the mixed system that rotates together with the container 1, the production cells 2 change the existing position with respect to the outside of the container with almost no change in the relative position in the liquid medium 3 due to the viscosity of the liquid medium 3, thus producing The cells 2 can be brought into a state in which gravity acts from a substantially different method, and the specific gravity and size of the cells 2, the specific gravity and viscosity of the liquid medium 3, the rotation speed of the container 1, etc. are appropriately selected. It can be realized by doing.

In such a state, looking at the production cells 2 in the mixed system, one production cell 2 is relatively indicated by the arrow A in the liquid medium 3 as schematically shown in FIG. It is presumed that the circular movement of the cells causes the production cells 2 to be uniformly dispersed in the liquid medium 3 by this motive force. This dispersion occurs extremely quickly depending on the conditions, and the production cells 2 are evenly dispersed in the axis X direction. Therefore, the production cells 2 are dispersed in the liquid medium 3 with an extremely high degree of dispersion. It can be dispersed. The reason for this dispersion in the direction of the axis X is not fully clarified, but it is presumed that it is because convection that moves so as to diffuse in the liquid medium 3 occurs, and further, this is due to the convection in the initial period immediately after the start of rotation. It is also considered that the thermodynamic nonuniformity of the distribution state of the producer cells 2 in the axis X direction is involved.

In the present invention, the reason why the axis of rotation of the container is required to be substantially horizontal is that when the axis has an angle with respect to the horizontal, the cells in the liquid medium are not uniformly dispersed in the container. This is because. In this case, the inclination of the axis which is allowed as being substantially horizontal may vary depending on the shape of the container, but for example, the inclination up to about 5 ° with respect to the horizontal is substantially horizontal. Can be tolerated,
The inclination is preferably within 3 ° with respect to the horizontal.

In any case, the present invention, as described above, does not disturb the liquid medium relatively, and gently moves the producing cells inside the liquid medium to uniformly disperse the producing cells. It can be brought into contact with a new liquid medium, and the dispersed production cells are not subjected to a shearing force or a force with a large impact, and thus the production cells are not damaged. The producing cells can be kept alive under suitable environmental conditions, and as a result, the physiologically active substance can be produced with high efficiency.

Incidentally, in the present invention, a special method is not required to fill the container with the liquid medium and the producing cells,
It suffices to fill the premixed liquid medium with the producing cells. However, when the production cells are adhesion-dependent cells, the step of adhering the adhesion-dependent cells to the carrier particles is more preferable than the case of filling the container with the carrier particles to which the adhesion-dependent cells are adhered in advance. By a similar operation using, by uniformly dispersing the carrier particles and the adhesion-dependent cells in the liquid medium without applying shearing force,
It is desirable to allow the production cells to adhere to the surface of the carrier particles evenly and efficiently in each carrier particle, in which case the present invention can be carried out subsequent to this adhesion step.

In order to keep the producing cells alive for a long time, it is necessary to change the liquid medium. Also in the present invention, it is necessary to replace the liquid medium in the treatment for a long period of time. The liquid medium can be replaced, for example, by stopping the rotation of the container to replace a part or the whole of the liquid medium, installing a liquid medium supply pipe and a discharge pipe in the container, and continuously supplying a new liquid medium during processing. And a method of discharging the old liquid medium at the same time. By supplying a new liquid medium in this way, it is possible to sufficiently maintain the survival of the producing cells for a long time, usually several days or longer.
If a new liquid medium is supplied through the supply pipe at all times as described above, the supply amount and the discharge amount per unit time may be small, so that the laminar flow of the liquid in the container,
It is possible to create a state in which a mechanical flow such as turbulent flow is not substantially generated, which prevents turbulence from occurring in the environment of the producing cells. In order to increase the production efficiency of the physiologically active substance, it is preferable to constantly supply the liquid medium through the supply pipe.

Further, in the case of producing a culture solution containing a physiologically active substance by adhesion-dependent cells according to the present invention, when the method of constantly supplying the liquid medium to the container through the supply pipe and constantly discharging it through the discharge pipe is used, the liquid to be supplied. A method of supplying carrier particles and adhesion-dependent cells together with a medium to a container, and extracting a part of the carrier particles adhering the physiologically active substance and / or adhesion-dependent cells produced together with the liquid medium discharged from the container. If used, the exchange of the liquid medium, the adhesion of the adhesion-dependent cells to the carrier particles, the survival maintenance, and the withdrawal of the culture solution containing the produced physiologically active substance can be continuously performed.

In the present invention, the carrier particles used in the case of utilizing adhesion-dependent cells may be those suitable for the adhesion and life support of adhesion-dependent cells, for example, synthetic polymers such as polystyrene, proteins and polysaccharides. Particles whose surface is formed by a natural polymer such as, but the carrier particles are convenient to have magnetism, by using a magnet to collect, move, treat the carrier particles. , Other handling can be performed easily and quickly. The carrier particles having magnetism include those obtained by binding magnetic substance powder with a polymer material, and those obtained by coating a magnetic core with a polymer material, and specific examples of the magnetic substance include iron, Examples thereof include cobalt, nickel, alloys thereof, low carbon steel, silicon steel, γ-iron oxide, ferrite, magnetite and the like. The specific gravity of the carrier particles varies depending on the viscosity and specific gravity of the liquid medium, but is generally within the range of 1.0 to 1.5.
The carrier particles preferably have a particle size of about 40 to 500 μm and preferably have a spherical shape, but may have a granular shape, a cylindrical shape or the like, or may have an irregular shape.

The seeding amount of suspension-proliferating cells or adhesion-dependent cells per 1 ml of liquid medium is generally 1 × 10 ⁴ to 1 × 10 ⁶ , and the amount of carrier particles used to utilize the adhesion-dependent cells is The number is usually about 1 × 10 ⁴ to 1 × 10 ⁷ per 1 ml of liquid medium.

In the present invention, it is desirable that the container is filled with a mixed system of the liquid culture medium and the producing cells in a filled state so that there is no space, thereby reliably rotating the mixed system together with the container. If there is only a small amount of space when it exists, the disturbance in the liquid medium due to the existence of the space is limited to a small area in the upper part of the mixing system in the container. It does not invalidate the effect of the present invention. However, if the ratio of the space in the container increases, the mixing system cannot be rotated together with the container without disturbing it, and the object of the present invention cannot be achieved. Therefore, in the present invention, the filling rate of the mixed system in the container is 98% by volume or more.

In the present invention, the rotation speed of the container in rotation cannot be unconditionally defined by the size and specific gravity of the producing cells, the viscosity of the liquid medium, the shape and size of the container, etc., but usually 5 to 50 r. pm, preferably 10-30 r.pm.
Adjust the rotation speed so that it is about the same level.

Further, the shape of the container is preferably a cylindrical shape, as in the above-mentioned example, but there is no problem even if it is a rectangular tube shape or a spherical shape, and the rotation method is essentially such that the upper and lower sides of the container are interchanged. It is sufficient that the container 12 is held at the tip of an arm 11 that rotates about a horizontal axis of rotation 10 as shown in FIG. 4 so that it moves circularly in the vertical plane. May be. In this case, the container 12 is rotated around the horizontal rotary shaft 10 in a state of being separated from the horizontal rotary shaft 10, but the same effect as the above can be obtained. Further, as shown in FIG. 5, the cylindrical container 20 may be rotated so as to rotate about a horizontal axis Y extending in its radial direction.

FIG. 6 shows an example of the present invention. In the example of this figure, a container is constituted by a bottom plate 30 and a bottomed cylindrical container body 31. That is, the container body 31 is pressed against the bottom plate 30 via the O-ring 33 by the holding mechanism 32 fixedly provided on the bottom plate 30, and the inner space thereof is used as a survival maintaining region of the producing cells. The bottom plate 30 is fixed to a rotating sleeve 35, and the rotating sleeve 35 is rotatably and horizontally held by a jacket member 38 held by a stand 37 via a dry bearing 36. The bottom plate 30 has an opening at the center, and this opening allows the passage of liquid but not carrier particles.
It is blocked by 40, and an inner conduit 41 passing through this mesh 40 is fixed to the mesh. The inner conduit 41 has an opening at its tip, extends through the inside of the rotary sleeve 35, and its rear end is rotatably connected to the supply pipe 51 from the outside at the connection seal portion 50 so as to communicate therewith. . An outer conduit 42 is fixed to the inner circumference of the rotating sleeve 35 so as to have a double-pipe structure with respect to the inner conduit 41, a tip end thereof is connected to communicate with an opening of the bottom plate 30, and a rear end thereof is a connecting seal portion.
A discharge pipe that is rotatable at 50 and extends to the outside
It is in communication with 52. Reference numeral 60 denotes a driven gear fixed to the rotary sleeve 35, which meshes with a drive gear 61 driven by a motor (not shown).

In the structure as described above, the driving gear 61 is driven to rotate the rotating sleeve 35, which causes the bottom plate to rotate.
The container constituted by 30 and the container body 31 is rotated around a horizontal axis, and at the same time, the inner conduit 41 and the outer conduit 42 are rotated together. Then, by filling the liquid medium and the producing cells into the container, the producing cells are uniformly dispersed in the container according to the above-mentioned procedure.

Thus, in this device, since the supply pipe 51 communicates with the inner conduit 41 and the outer conduit 42 communicates with the discharge pipe 52, these allow the container to be rotated to disperse the production cells and stabilize the production. It is possible to supply a new liquid medium into the container and discharge a corresponding amount of the used old nutrient-depleted liquid medium to the outside while ensuring a survival maintaining environment. Then, since the production cells are prevented from being discharged, the production cells can be kept alive for a long period of time.

FIG. 7 is an example of a system chart for maintaining the survival of producing cells according to the present invention, in which 70 is a liquid medium tank, 71 is a container, 72 is a liquid medium discharge tank, and a container is provided by a monitor mechanism 73. For the liquid medium immediately after being discharged from 71, for example,
And the amount of dissolved oxygen detected by the DO sensor 75, and the amount of new medium supplied to the container 71 is regulated by the control valve 76 according to the result. Reference numerals 77 and 78 respectively denote a pH sensor and a DO sensor provided in the liquid culture medium tank 70. The alkali is added from the alkali tank 80 at a controlled ratio according to the pH of the liquid culture medium measured by the pH sensor 77 to adjust the pH. Is adjusted to extend into the liquid medium tank 70, for example, 5
The air supply amount is controlled in accordance with the detection result of the DO sensor 78 via a control valve 82 inserted in an air supply pipe 81 that supplies air containing carbon dioxide gas.

According to such a system, it is possible to always maintain a good environment for maintaining survival in the container, and it is possible to uniformly disperse the producing cells in a liquid medium at a high degree of dispersion and to maintain the survival. It is possible to produce a culture solution containing a physiologically active substance secreted from early cells with high efficiency on an industrial scale.

8 and 9 show another example of the apparatus used in the method of the present invention, in which a horizontally extending rotary shaft 102 is provided in an outer casing 101 having a heat insulating material layer, and One end is connected to a motor 103 arranged outside the outer casing 101 via a power transmission mechanism 104. A plurality of (four in the illustrated example) arms 105 that radially project are fixed to the rotating shaft 102, and a holder 107 that detachably holds a cylindrical container 106 is provided at the tip of each arm 105. ing. A heater (not shown) is provided inside the outer casing 101.

According to the apparatus having such a configuration, the plurality of containers 106 are filled with the liquid culture medium and the production cells, and the rotating shaft 102 is rotated to rotate the containers 106 in the same manner as described above.
Inside, the production cells can be suitably and uniformly dispersed in a liquid medium to keep them alive.

Specific examples of physiologically active substances and producing cells that can be suitably obtained by applying the present invention are shown in Table 1.

The liquid medium used in the present invention is not particularly limited, and a known medium can be used as it is, for example, RPMI 1640 medium, Eagle MEM medium, Dulbecco's modified Eagle medium, Earle medium 199, Ham F1.
2 media and the like. These media include 5
It is preferable to add and use, for example, fetal bovine serum, newborn bovine serum, horse serum, human serum at a ratio of -10% by volume. It is also possible to use a serum-free medium that does not use serum, such as HB102 (Hana Biologics), RITC55-9 medium and the like. The specific gravity of the liquid medium is generally 1.00 to 1.05. Note that it is usually necessary to dissolve oxygen and carbon dioxide in the liquid medium, and the production and maintenance of the producing cells are usually performed at 30 to 40 ° C, preferably 36 to 37 ° C.

〔Example〕

Examples of the present invention will be shown below, but the present invention is not limited thereto.

Example 1 In a container having an internal volume of 300 ml of the apparatus having the configuration shown in FIG. 6, 5% by volume of newborn calf serum and crosslinked dextran microcarriers having a diethylaminoethyl group (diameter 180
˜220 μm) at a rate of 3 g / ml in Eagle MEM medium at a rate of 1.2 × 10 ⁵ cells / ml of human normal diploid cells, and the mixture is filled with air so that the temperature is 37
The temperature of the mixed system was adjusted to 7.2 by adding the Eagle MEM medium containing oxygen and carbon dioxide gas successively while rotating the container at a speed of 12 rpm at 144 ° C. for 144 hours.
As a result, the cell number was 3 × 10 ⁶ cells / ml.

Next, Eagle MEM medium containing 100 IU / ml interferon and carboxymethyl cellulose was used as the medium.
It is supplied at a rate of 0 ml / hr into a container and the temperature is 37 ° C and the pH is 7.2.
The container was rotated for 4 hours. Then, the continuous feeding of the medium was stopped, 10 μg / ml of cycloheximide was added to the container, and rotation was continued at 37 ° C for 4 hours, then 4 μg / ml of actinomycin D was added, and the mixture was rotated at 37 ° C for 1 hour. Let Then Eagle MEM containing 0.5 g / methylcellulose
The medium was continuously supplied at a temperature of 37 ° C. so that the mixed system in the container had a pH of 7.2, and the medium was rotated for 48 hours. As a result, the cell number was 4.8 × 10 ⁶ cells / ml, and the interferon titer was 35.
It was 000 IU / ml.

Comparative Example 1 A spinner flask having an internal volume of 500 ml was used, the amount of the medium was 300 ml, and the operation was performed under the same conditions as in Example 1. As a result, the final cell number was 1.6 × 10 ⁶ cells / ml. , The interferon titer was 27,000 IU / ml, and the production efficiency was very low.

Example 2 A mixed system in which a mouse / mouse hybridoma CIOB 6 strain was inoculated in a serum-free ITES medium at a rate of 4 × 10 ⁴ cells / ml in a container having an internal volume of 300 ml of the apparatus having the configuration shown in FIG. To prevent the entry of air, and under the environment of a temperature of 37 ° C, while rotating the container at 12 rpm for 200 hours, ITES medium in which oxygen and carbon dioxide were dissolved was added successively to adjust the pH of the mixed system. As a result of maintaining at 7.2, the cell number was 1.2 × 10 ⁷ cells / ml, and 0.8 mg of the monoclonal antibody was produced per 1 ml of the medium.

Comparative Example 2 A spinner flask having an internal volume of 500 ml was used, the amount of the medium was 300 ml, and the operation was performed under the same conditions as in Example 2 (however, the rotation speed was 7 rpm). As a result, the number of cells was 5 × 10 ^6. / M
The production amount of the monoclonal antibody was as small as 0.2 mg per 1 ml of the medium.

Example 3 140 mM sodium chloride, 54 mM potassium chloride, 1
10 ^{3 of} human renal tumor cells and lymphoblastic Namalwa cells each obtained by excision from a renal tumor patient excised and dissected into a saline solution containing mM sodium monophosphate and 2 mM calcium chloride / It was suspended to a volume of 1 ml and mixed with the same salt solution containing Sendai virus previously inactivated by UV light under ice cooling and the temperature was raised to 37 ° C after 5 minutes. Human erythropoietin producing ability was introduced into lymphoblastic Namalwa cells.

The above-mentioned lymphoblastic Namalwa cells were seeded at a rate of 5 × 10 ⁴ cells / ml in an Earle medium 199 containing 10% by volume of fetal bovine serum in a container having an internal volume of 300 ml of the apparatus having the configuration shown in FIG. The system was filled to prevent air from entering, and Earle medium 199 in which oxygen and carbon dioxide were dissolved was added successively while rotating the container at 12 rpm for 200 hours in an environment of temperature 37 ° C, and the pH of the mixed system was adjusted. Was maintained at 7.2. as a result,
The number of cells was 1.5 × 10 ⁷ cells / ml, and 150 units of human erythropoietin was produced per 1 ml of the medium. The unit here is one of the international standard products distributed by the World Health Organization (WHO).
It is 1/10 the activity contained in the ampoule.

Comparative Example 3 A spinner flask having an internal volume of 500 ml was used, the amount of the medium was 300 ml, and the operation was performed under the same conditions as in Example 3 (however, the rotation speed was 7 rpm). As a result, the cell number was 4.8 ×. 1
0 ⁶ cells / ml, and the human erythropoietin production of Chin been filed those 10 units and less per medium 1 ml.

Example 4 10.9 g of RPMI-1640, 60 mg of kanamycin sulfate, N-2-hydroxyethylpiperazine-N'- in a container having an internal volume of 300 ml of the apparatus having the configuration shown in FIG.
2-ethanesulfonic acid 1.19 g, sodium hydrogen carbonate 1.05
g, transferin 1 mg, ethanolamine 4.6 mg, and selenite 13 μg were inoculated with a mixed system in which QG-90 cells were inoculated at a rate of 2 × 10 ⁴ cells / ml, and the mixture was filled so as not to enter air. The temperature of the mixture was kept at 37 ° C. for 240 hours, while the container was rotated at 12 rpm, oxygen and carbon dioxide were successively added to maintain the pH of the mixed system at 7.4. As a result, the number of cells was 7.4 × 10 ⁶ / ml, and the titer of Tesiu plasminogen activator was 25I.
It was U / ml (urokinase conversion).

Comparative Example 4 A spinner flask having an internal volume of 500 ml was used, the amount of the medium was 300 ml, and the operation was performed under the same conditions as in Example 4 (however, the rotation speed was 7 rpm). As a result, the cell number was 1.2 × 10 ^6. The number of Teshiyu plasminogen activator was as low as 2 IU / ml.

Example 5 10% by volume of fetal bovine serum and 1.0 g of L-glutamine were placed in a container having an internal volume of 300 ml of the apparatus having the structure shown in FIG.
Cytodex 3 3 g / in Eagle MEM medium containing
1 x 10 cells of human embryonic kidney (Microbiological Associates)
Seeding was performed at a rate of ⁵ cells / ml. Fill the mixing system so that air does not enter, and continue to add the above-mentioned Eagle MEM medium in which oxygen and carbon dioxide are dissolved while rotating the container at 15 rpm for 100 hours in an environment of a temperature of 37 ° C. and mixing. The system pH was maintained at 7.4. Then 1 g of fumaric acid
/ And lactalbumin hydrolyzate 5g /
Earle medium 199 is supplied to the container at a rate of 300 ml / hr and the temperature is 37
The vessel was maintained at pH 7.4 for 250 hours at 15 rpm while rotating at 15 rpm. As a result, the urokinase titer was 380.
IU / ml.

Comparative Example 5 A spinner flask having an inner volume of 500 ml was used, the amount of the medium was 300 ml, and the operation was performed under the same conditions as in Example 5 (however, the rotation speed was 60 rpm). As a result, the titer of urokinase was 200 IU. It was as low as / ml.

〔The invention's effect〕

INDUSTRIAL APPLICABILITY The present invention satisfactorily disperses the production cells without applying shearing force to the mixed system of the production cells and the liquid medium, and maintains the survival of the production cells in a stable state without mechanically vigorous movement. This can result in
It is characterized in that the production cells can be actively maintained for survival and the physiologically active substance can be produced with high efficiency, and that the physiologically active substance can be advantageously produced even in large-scale implementation. In the practice of the present invention, growth of producer cells usually occurs in parallel, which is preferred because of increased efficiency. However, the growth of the producing cells is basically irrelevant, and even if the growth rate is low or extremely low, it was secreted from the cells with high efficiency by setting suitable conditions in the present invention. A culture solution containing a physiologically active substance can be produced.

It should be further noted that the physiologically active substance produced according to the present invention has a feature that its potency is high, and therefore from the viewpoint of producing the physiologically active substance,
Very high efficiency is obtained.

[Brief description of drawings]

1 and 2 are perspective views and cross-sectional views for explaining one example of the present invention, FIG. 3 is an explanatory view for the movement of producing cells in a liquid medium, and FIGS. 4 and 5 are the present invention respectively. An explanatory cross-sectional view and a perspective view showing another example,
FIG. 6 is a cross-sectional view showing an example of an apparatus for carrying out the method of the present invention, FIG. 7 is an example of a system chart for carrying out survival maintenance of producing cells according to the present invention, and FIGS. 8 and 9 are It is a longitudinal side view and front view for explanation showing other examples of the device used for the method of the present invention. 1,12,20 …… Container, 2 …… Production cell 3 …… Liquid medium, 30 …… Bottom plate 31 …… Container body, 32 …… Pressing mechanism 35 …… Rotating sleeve, 36 …… Dry bearing 37 …… Stand , 38 ...... Outer member 40 ...... Mesh, 41 ...... Inner conduit 42 ...... Outer conduit, 50 ...... Connection seal part 51 ...... Supply pipe, 52 ...... Discharge pipe 60 ...... Drive gear, 61 ...... Drive gear 70 …… Liquid culture medium, 71 …… Container 72 …… Discharge tank, 73 …… Monitor mechanism 74, 77 …… pH sensor, 75, 78 …… DO sensor 80 …… Alkaline tank, 81 …… Air pipe 101… … Outer casing, 102 …… Rotary shaft 103 …… Motor, 104 …… Power transmission mechanism 105 …… Arm, 107 …… Holder

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Claims (1)

[Claims] 1. A mixed system consisting of liquid medium and particles to be dispersed to which adhesion-dependent cells producing a physiologically active substance are adhered or suspension-proliferating cells producing a physiologically active substance is filled at a volume ratio of 98% by volume or more. Rotate the culture container filled so that it rotates about a substantially horizontal axis, or
Alternatively, the mixed system is rotated together with the culture vessel by rotating the culture vessel around the axis in a state of being separated from the substantially horizontal axis, whereby the particles to be dispersed or floating growth in the mixed system. Cells, wherein the cells to be dispersed are dispersed in the liquid medium to thereby keep the adhesion-dependent cells or suspension-proliferating cells alive. A method for producing a culture solution containing a physiologically active substance secreted from the culture medium.