JPH06225758A - Process for culturing anchor-dependent cells and culture system - Google Patents

Abstract

PURPOSE:To continue the proliferation of a large volume of cells in the cultivation tank by allowing the culture mixture to flow from the upper-stream chamber to the down-stream chamber as the microcarrier is separated into individual chambers in the cylindrical cultivation tank to feed the oxygen through the oxygen-permeating hollow fiber membranes into the culture mixture. CONSTITUTION:The cylindrical cultivation tank 1 is separated with partition walls having oxygen-permeating hollow fiber membrane as a mid-layer 8 and the individual chambers 10 between the walls are filled 95% by volume with microcarrier 11. The cultivation tank is kept at a constant temperature with thermostated water and the culture mixture 19 is allowed to flow in the arrow direction, as oxygen is fed through the hollow fiber membranes and the tank is rotated alternately in one and the other directions to fluidize the microcarrier bed. When valve 22 is contracted, the microcarrier moves between the chambers and a part of the microcarrier is taken out of the tail chamber, while the make-up microcarrier inoculated with seed cells is introduced into the chamber of the upper-most stream.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and apparatus for culturing adherent animal cells, and more particularly to a method and a method suitable for efficiently and continuously culturing adherent animal cells in a culture carried on a microcarrier. Regarding the device.

[0002]

Perfusion culture of adherent animal cells is one of the most powerful means for producing useful cell secretory components. Among them, the microcarrier method is suitable for culturing adherent cells on a practical scale. In order to increase the production efficiency of this method, it is necessary to improve the filling rate of the carrier in the culture tank and to supply oxygen that can follow it.

The microcarrier method is roughly classified into a fluidized bed method for keeping the carrier in a floating state and a fixed method for filling the carrier in a force ram or the like. The latter can keep the filling rate high, but the carrier bed is likely to be clogged with cell growth and the oxygen supply to the bed is likely to be insufficient. On the other hand, the former is easy to avoid blockage and is relatively easy to deal with oxygen supply.

The present inventor has previously proposed a culture method and apparatus in which a rotary horizontal cylindrical tank is filled with a carrier and a medium is supplied to grow cells on the surface of the carrier (Japanese Patent Application No. 3-
232968 specification).

[0005]

This method is a very useful method in that it enables smooth mixing in the tank without using a mechanical seal which has a risk of invading various bacteria as in the past. In this tank, the carrier bed is moved from the upstream side to the downstream side through the overflow plate so that the carrier can be renewed. At this time, since the oxygen supply and sensors are arranged on the horizontal rotation axis, the overflow weir must be arranged below these elements. Therefore, the carrier filling rate can be made higher than in the past, but was at most 40%. An object of the present invention is to provide a method for culturing in a rotary culture using a microcarrier at a higher carrier filling rate and under a supply of oxygen, and a culture apparatus therefor.

[0006]

The inventor of the present invention has reached the present invention as a result of earnest efforts to improve the drawbacks of the prior art.

The first feature of the method according to the present invention is that the microcarrier bed is vertically divided into a plurality of chambers, and the ratio of the bed volume to the total volume of the chambers is at least 50% or more and 95% or more.
Keep the following, fill the remaining space with the medium, in a state where the microcarriers are isolated in each chamber, let the medium pass from the most upstream chamber to the downstream chamber, at which time the medium is sequentially passed into each chamber. This is repeated by bringing the membrane into contact with an oxygen-permeable hollow fiber membrane through which an oxygen-containing gas is passed and then passing the membrane through the bed.

The second feature of the method according to the present invention is that while the medium is refluxing, the bed is repeatedly rotated once around the horizontal axis for at least one rotation and then reversely rotated to cause the bed to flow, and the space between the chambers is repeated. With the microcarrier bed connected, part of the used microcarrier bed in the most downstream chamber is extracted, fresh microcarriers with seed cells attached are introduced into the most upstream chamber, and the outer jacket thermostatic water outside the tank is used. It is to circulate and keep warm.

Further, the third feature of the method of the present invention is that
To construct a process and system that incorporates a carrier renewal operation into the culture described above. That is, the extracted microcarriers are mixed with at least a carrier-decomposition-containing solution at a constant temperature and under oxygen supply to dissolve the carrier, and the cells are dispersed in the solution to obtain an enzyme-treated cell dispersion liquid, and the enzyme-treated cell dispersion liquid is obtained. The solution is centrifuged to separate the cells into the used enzyme solution, the used enzyme solution is taken out of the system, the medium and fresh microcarriers are added to the collected cells, and the mixture is mixed under constant temperature and enzyme supply. Cells are adhered to fresh microcarriers to obtain cell-adhered microcarriers, and the microcarriers are transferred to the uppermost chamber.

The main features of the culture device according to the present invention are that the rotation axis is in the lateral direction, it has a cylindrical shape, and flexible pipes that can rotate for one rotation or more are connected to the side surface, and a plurality of insides of the tank are provided. It is vertically partitioned into a plurality of chambers by a disk-shaped partition, and the partition has a bag-like shape consisting of two disks, and has a through hole that communicates with the chambers on both sides at the center of the partition wall. There is a microcarrier-impervious liquid-permeable mesh hole near the center or the circumference of the disk on the side, and the disk on the downstream side is impermeable to the microcarrier toward the circumference or toward the center. It has a hole with a liquid-permeable net, an oxygen-permeable hollow fiber membrane is placed between two discs, and an opening / closing valve that can be operated from the outside is provided in the through hole at the center of the partition wall. It has a constant temperature water jacket on the outer surface of the tank and a drive mechanism for rotating the tank back and forth.

A fourth feature of the device according to the present invention is that it is a cell culture system characterized by comprising at least the following elements. That is, a medium circulating means through the medium inflow port and the medium outlet of the culture tank through a contact portion with the D0 sensor and the pH sensor, a constant temperature water circulating means between the circulation constant temperature tank and the culture tank jacket, and gas mixture. Adjusting and mixing means, means for venting mixed gas to the oxygen permeable membrane, medium storing and supplying means, used medium extracting and storing means, at least a horizontally rotatable ball rotor and rotation of the rotor A liquid transfer pipe having an open end close to the inner bottom surface of the rotor on the axis and a blade capable of moving up and down,
In addition, it is composed of a centrifugal separation means which stores these in a pressure resistant chamber.

The cells applicable to the present invention are not particularly limited as long as they are animal cells having adhesiveness. As the carrier, a non-protein organic material such as cellulose, hemicellulose, crosslinked dextran, or a derivative thereof is used.

The culture tank has a cylindrical shape arranged laterally, and the inside is divided into a plurality of chambers by a plurality of disk-shaped partition walls. The volume of the carrier floor is at least 5 of the volume of each chamber
It occupies 0% or more and 95% or less, preferably 80 to 95%, and the remaining space needs to be filled with a medium. Rolling is performed by repeating forward rotation for one rotation or more about the tank rotation axis and then reverse rotation for the same rotation speed. The rotation speed is set to a rotation speed at which the flexible pipes or wirings connected to both ends of the tank do not lose their functions due to blockage or crossing.

The partition walls are two discs which are parallel and maintain a narrow gap, and have a structure in which a short tube is adhered to a hole in the center of both discs. Furthermore, the plate on the upstream side has holes that are netted toward the circumference or near the center, and the disk on the downstream side has holes that are netted toward the center or near the periphery. These holes may be small circles or annular shapes. The mesh has a pore size that allows passage of the medium smoothly but does not allow passage of microcarriers. The pore size depends on the particle size of the carrier used, but generally 70 to 90% of the carrier particles, that is, 50
Is performed in the range of up to 200 μm. An oxygen-permeable hollow fiber membrane is incorporated in the gap between both plates. A porous polymer film is usually used as the film. The membrane is monolayered or multilayered so as to increase the contact area with the medium, and is modularized at a packing density that does not significantly hinder the passage of the medium. The hollow fiber membranes between the chambers are connected in series or in parallel and the oxygen-containing gas is aerated. Further, the hollow fiber membrane is connected to a flexible pipe at the entrance and exit of the tank. The hole in the center of the partition is closed by a valve that can be operated from the outside during the reflux of the medium, and the carrier is kept isolated in each chamber. When updating the carrier, after extracting a part of the carrier in the most downstream chamber, each valve in the hole of the partition is opened by external operation, and the carrier corresponding to the amount of extraction moves to the downstream side and at the same time from the upstream side. Transfer fresh carrier with attached cells. The medium that came out of the culture tank was D0 and p
After coming into contact with the H sensor, it is returned to the upstream of the culture tank. Both the D0 sensor and the pH sensor are electrically connected to the flow rate control valve for oxygen, air, and carbon dioxide by each controller. As the medium components are exhausted, a part of the used medium is extracted and replenished with an equal amount of fresh medium.

The carrier extracted from the culture tank is introduced into the ball type rotor of the centrifuge, the enzyme solution is added through a fixed pipe extending from the rotor, and the vertically movable blades are lowered and fixed below the liquid surface. The carrier is enzymatically decomposed by slowly rotating the rotor for a predetermined time. The carrier is enzymatically decomposed by rotating the rotor at a slow speed.
After stopping the rotor, the rotor is rotated at a speed at which cells settle while the blades are pulled above the liquid surface and centrifuged to deposit cells on the inner wall of the rotor. After stopping the rotor, the used enzyme solution is removed to the outside of the system through a fixed pipe that opens near the bottom of the rotor. If necessary, in order to completely remove the used enzyme solution adhering to the inside of the rotor and the cells, introduce the medium, lower the blade and rotate slowly, raise the blade and centrifuge, and then remove the supernatant. You can insert a wash operation to remove.

[0016]

By dividing the carrier bed into independent chambers when the medium is refluxed in this manner, the carrier can be retained at a high filling rate during reflux, and the productivity can be improved accordingly. Further, the two discs of the partition wall allow the medium and the hollow fiber membrane to be efficiently contacted with each other to smoothly dissolve oxygen in the medium. Moreover, since the carrier beds are arranged in series with the oxygen-permeable hollow fiber membrane module sandwiched between them, sufficient oxygen can be supplied to the highly-filled carrier beds, and a short circuit between the carrier and the medium can be prevented. Further, the extracted carrier can be automatically renewed under no condition by a series of operations using a centrifugal separator.

[0017]

Embodiments of the present invention will be described with reference to the drawings.

(Embodiment 1) FIG. 1 and FIG. 2 are sectional views of a culture tank of the present invention, and FIG. 3 is a system diagram showing the structure of a culture system.

The culture tank 1 is made of stainless steel and has a cylindrical shape with an outer diameter of 200 and a length of 400 m, which is horizontally held by a support 7 and a bearing 2 and is rotatable back and forth. Rotation is performed by the motor 3, the transmission 4, and the gear 5, and forward and reverse rotation is possible by switching the wiring of the motor. The inside of the tank is surrounded by an outer jacket 7 in which constant temperature water 6 circulates from the outer wall, the inside of the tank is divided by partition walls 9 sandwiching an oxygen permeable hollow fiber membrane 8, and microcarriers with a particle size of 200 μm are provided in each chamber 10 between the partition walls. 11 is filled. The hollow fiber membranes 8 are polytetrafluoroethylene tubes having an outer diameter of 3 mm and an inner diameter of 1.5 mm and a pore size of 0.1 μm, and the hollow fiber membranes 8 are connected in series from the upstream portion to the downstream portion. The disc 12 on the upstream side of the partition wall has a C-
As shown in the C ′ cross section, it has a hole 14 formed by stretching a stainless steel net having a hole diameter of 100 μm from the circumference, and has a downstream side circular plate 15
Has a hole 17 of a net 16 having the same hole diameter from the center as shown in the section AA '. The upstream side disk 12 and the downstream side disk 15 are joined to each other via a short pipe 18 at the central portion and kept parallel to each other. As shown in the BB ′ cross section, the hollow fiber membrane 8 is accommodated between the two disks in a bent state by the hollow fiber membrane support 24 within a range where the reflux of the medium cannot be prevented. The medium 19 that has flowed in from the upstream side efficiently contacts the hollow fiber membranes 8 through the path indicated by the arrow in the figure to dissolve oxygen in the medium. In each hole 20 at the center of the partition wall, a valve 22 composed of an expander, which is large enough to fit in the hole 20 and is connected in series by a pressurized air pipe 21, is provided. The expansion of the valve 22 blocks the movement of the carrier 11 to the adjacent chamber. The medium 19 enriched with oxygen in the hollow fiber membrane 8 is the carrier bed 11
Oxygen and nutrients are supplied to the cells while passing through the cell, and the hollow fiber membrane 8 in the next partition wall is again enriched with oxygen. The cells proliferate on the surface of the carrier, and when the surface is saturated, the proliferation stops,
Then, after continuing to secrete the active ingredient for a certain period of time, the cells age and die. At the stage where cells enter senescence, a part of the carrier 23 to which the senescent cells are adhered is pulled out of the carrier and the pipe 2
The cells are extracted from the downstream side after passing through 5, and the cells are reused as seed cells at the time of carrier renewal described later.

The circulating water in the jacket is constantly circulated through a circulating constant temperature water tank 26 as shown in FIG. The culture medium 19 discharged from the most downstream side of the culture tank 1 is circulated by a pump 31 via a culture medium circulation pipe 27 and a sensor storage tank 28. A D0 sensor 29 and a pH sensor 30 are arranged in the sensor storage tank 28 to detect D0 and pH, and the D0 and pH controller 3
2, the gas filters 34, 3 are set so as to approach the set value.
Aseptically treated through 5, 36, oxygen 42, carbon dioxide 4
3, the flow rate ratio of the air 44 to the valves 36, 37, 38, 3
The oxygen-containing gas 25 adjusted by 9, 40 is communicated with the hollow fiber membrane 8. The exchange of the medium is carried out based on the set exchange rate, and a part of the medium discharged from the downstream of the culture tank 1 is guided by the process sequencer 45 to the unused medium 47 by opening the valve 46 and stored in the unused medium storage tank 48. At the same time, the fresh medium 19 is supplied from the medium storage tank 49 by the pump 50 via the valve 51 to the uppermost stream of the culture tank 1. The extracted carrier 23 is introduced into the rotor 57 in the centrifugal separator 54 through the valve 50, the transfer pipe 55, the valves 52 and 53, and the liquid supply nozzle 56. The rotor is in a rotor chamber 55 composed of a pressure resistant wall 58 with a constant temperature water circulation jacket 59, is supported by a bearing 62 in the rotor chamber, and can be rotated by a rotation speed adjusting motor 61 via a permanent magnet 60. By sending the pressurized air 44 to the bellows 64 via the air pipe 66 to the upper wall of the rotor chamber 55, or by expanding and contracting the bellows 64 by opening the valve 69 and discharging the air in the bellows 64, A baffle 62 capable of moving only up and down inside the rotor is connected via a support rod 65. Further, there is a liquid transfer pipe 63 penetrating the upper wall of the rotor chamber and having a lower end opened to the center of the inner bottom surface of the rotor, and the outside of the centrifuge 58 is branched to a pipe 70 and a pipe 67. The pipe 70 is connected via a valve 71 and a pump 72 to an enzyme treatment waste liquid storage tank 74 that stores an enzyme treatment waste liquid 73. The other pipe 67 is connected to the carrier floor introduction pipe 75 of the culture tank 1 via the valve 68.
Connected to. The pipe 76 further branches into pipes 77 and 55. The pipe 77 is connected via a valve 79 to an enzyme liquid storage tank 82 that stores an enzyme liquid 81, and the pipe 55 branches into a pipe 78 before the pump 51, and a medium suspension of a fresh carrier 83 via a valve 80. It is stored in a fresh carrier storage tank 84 which stores 83. Valves other than the valves 37, 38, 39 and all pumps and motors 61 and 3 are wired so that they are operated by a program preset by the sequencer 45.

Example 2 A spherical porous carrier made of cellulose as a microcarrier (diameter 100 to 100)
200 μm), and CHO cells belonging to the adherent cells were cultured by the device of Example 1 using a Dulbecco's modified Eagle MEM medium containing 10% bovine serum as a medium.

After the whole system was sterilized with steam and cooled, the medium 19 was supplied from the medium storage tank 49 into the culture tank 1 by the pump 50 to fill the inside. Next, the valve 60 is opened to allow the air in the pipe 21 and the valve 20 to escape to connect the chambers in the culture tank 1, and then the fresh carrier is stored in the fresh carrier storage tank 84 by the pump 51 via the pipes 78 and 67. It was sent to the chamber and filled up to 90% of the effective volume in the tank. Circulating constant temperature water was circulated through the jacket 7 to equilibrate the inside of the tank to 37 ° C., and then aseptically through the pipe 83, 100 ml of the medium suspension of CHO cells (1 × 10 ⁴ cells / cell).
(Ml) was introduced at 10 ml / min. The oxygen-containing compound gas 25 is passed through the aerial fiber membrane 8 to obtain D0 and pH.
The controller 32 adjusted D0 to 1 ± 0.2 ppm and pH to 7 ± 0.1 ppm, and the pump 31 circulated the medium in the culture tank at 100 ml / min. The medium was circulated for 3 hours, and it was confirmed that the cells adhered to the carrier and the outflow of cells from the downstream of the tank was 5% or less. Continue circulating for 2 days as it is, and after confirming that cells have grown in all the beds, open valves 50, 52 and 53, and pipe 25,
A part of the carrier bed in the downstream chamber was taken out into the rotor 51 through 76, 55, 56 by the pump 51, and 0.7 liter corresponding to 0.5 chamber was extracted and introduced into the rotor having an effective volume of 1 liter. Then, 50 ml of an enzyme solution prepared by dissolving 5 × 10 ⁴ units of the filamentous fungus belonging to the genus Trichoderma and 30 units of trypsin in 0.05M phosphate buffer (pH 7.0) was added. Pipe 1 for bellows 40
Baffle 39 was dropped into the rotor by introducing air through 0, the rotor was stirred at 10 rpm and reacted at 37 ° C. for 10 minutes to dissolve the carrier to obtain a suspension of cells. Next, the valve 69 was opened and the air in the bellows 64 was evacuated to pull up the baffle 62 above the liquid surface, and then the rotor 57 was rotated at 800 rpm for 5 minutes to centrifuge the cells and deposit them on the inner wall of the rotor. After the rotor 57 stops rotating,
The supernatant enzyme treatment waste liquid 73 was discharged into the enzyme treatment waste liquid storage tank 74 by the pump 72 via the pipe 63 and the valve 71.
Next, from the fresh carrier storage tank 84, the medium suspension slurry of fresh carrier is withdrawn by the valve 8 in an amount equal to the amount taken out from the culture tank.
0, pipe 78, valve 53, nozzle 56 into the rotor 57, air is introduced into the baffle 64 to lower the baffle 62 below the liquid surface, and the rotor is rotated at 10 rpm to disperse the cell carrier suspension. Let Meanwhile, piping 6
7. The enzyme-containing gas was passed through the rotor at a rate of 2 ml / min through the valve 68 and stirred for 4 hours while oxygen was supplied to the cells to adhere the cells to the surface of the carrier particles.
The cell adhesion carrier suspension in the rotor is piped 63, 67,
It was introduced upstream of the rotor chamber through a valve 68 and a pipe 75. It was confirmed that the above-mentioned carrier was taken out, the cycle of introducing the cell-adhering carrier was carried out twice a day, and the culture was continued for 7 days to reach a level of cell growth. Meanwhile, the culture medium in the tank was exchanged once a day by carrying out the operation of exchanging a quarter of the culture medium in the tank with a fresh medium four times a day.

As a result of measuring the number of living cells adhering to the carrier extracted from the downstream, the cells in the carrier in the culture tank reached a high concentration state of 9 × 10 ⁶ cells / milliliter per effective volume of the culture tank. . This cell concentration is 9 times higher in cell productivity than the conventional roller bottle type in which the carrier filling rate is 10% or less.

[0024]

INDUSTRIAL APPLICABILITY According to the present invention, when adherent animal cells are perfused by the microcarrier method, they can be gently fluidized with a high carrier filling rate and correspondingly provide oxygen and nutrients necessary for growth. Therefore, a large amount of cells can be proliferated and maintained in the culture tank, and the productivity of useful secretory components can be improved.

[Brief description of drawings]

FIG. 1 is a cross-sectional view of a culture tank according to the present invention.

FIG. 2 is a sectional view of a culture tank according to the present invention.

FIG. 3 is a systematic diagram of a system and a flow according to the present invention.

[Explanation of symbols]

1 ... Culture tank, 7 ... Jacket, 8 ... Oxygen permeable hollow fiber,
10 ... Microcarrier floor, 26 ... Constant temperature water circulating water tank, 2
9 ... D0 sensor, 30 ... pH sensor, 32 ... D0 and p
H controller, 48 ... Used medium storage tank, 49 ... Medium storage tank, 57 ... Rotor, 62 ... Baffle, 64 ... Bellows, 74 ... Enzyme treatment waste liquid storage tank, 82 ... Enzyme liquid storage tank, 84
… Fresh carrier storage tank.

Claims (6)

[Claims] 1. A method of culturing adherent animal cells having the following characteristics by a microcarrier system. (1) The microcarrier bed is vertically divided into a plurality of chambers, and (2) the ratio of the volume of the microcarrier bed to the total volume of the chamber is 95.
% Or less, the remaining space is filled with the medium, and (3) the medium is passed from the most upstream chamber to the downstream chamber while the microcarriers are isolated from each chamber, and the oxygen-containing gas is sequentially supplied to each chamber. Repeatedly contacting with the oxygen permeable hollow fiber membrane passing through and then passing through the bed, and (4) during that time, the bed is fluidized by rotating the bed around the horizontal axis for one or more times in the forward direction and then in the reverse direction. (5) With the microcarrier bed between the chambers connected to each other, a part of the used microcarrier bed of the most downstream chamber is extracted, and fresh microcarriers having the seed cells attached are introduced into the most upstream chamber, ( 6) Circulate constant temperature water in the outer jacket outside the tank to keep it warm. 2. The method for culturing adherent animal cells according to claim 1, further comprising the following steps. (1) The extracted microcarriers are mixed with at least a carrier-degrading enzyme-containing solution at a constant temperature and under oxygen supply to dissolve the carrier, and the cells are dispersed in the solution to obtain an enzyme-treated cell dispersion liquid (( 2) Centrifuge the enzyme-treated cell dispersion to separate the cells and the used enzyme solution, (3) extract the used enzyme solution out of the system, and (4) add the medium and fresh microcarriers to the recovered cells. Then, mix under constant temperature and enzyme supply,
The cells are adhered to the fresh microcarriers to obtain the cell adhesion microcarriers, and (5) the cell adhesion microcarriers are transferred to the most upstream chamber. 3. The method for culturing animal cells according to claim 1 or 2, wherein the medium that has passed through the microcarrier bed is circulated, and a part of the circulating medium is replaced with a fresh medium. 4. The method according to claim 1, 2, or 3, wherein the used enzyme solution is extracted from the system, suspended in a fresh medium, the cells are centrifuged to wash the cells, and the cells are dispersed again in the fresh medium. A method of culturing animal cells, wherein the cell dispersion is centrifuged again, and then fresh microcarriers and fresh medium are added to bring the cells into contact with the microcarriers. 5. A cell culture device having the following features. (1) The axis of rotation is in the lateral direction, (2) it has a cylindrical shape, (3) flexible piping that can rotate more than one rotation is connected to the side surface, and (4) a plurality of discs inside the tank. Is partitioned into a plurality of chambers in the vertical direction by (5), and (5) has a partition having the following structure. (A) It has a bag-like shape consisting of two discs, (b) has a through hole that communicates with the chambers on both sides at the center of the partition wall plane, and (c) is closer to the center or the circumference of the disc on the upstream side. There is a microcarrier-impervious liquid-permeable mesh hole close to (d) the disk on the downstream side has a microcarrier impermeable liquid-permeable mesh stretched toward the circumference or toward the center. (E) An oxygen-permeable hollow fiber membrane is placed between two discs having a hole. (6) An opening / closing valve that can be operated from the outside is provided in a through hole at the center of the partition wall, (7) A constant temperature water jacket is provided on the outer surface of the tank, and (8) A drive mechanism for rotating the tank back and forth. 6. A cell culture system comprising at least the following elements. (1) A means for circulating a medium through the medium inlet and the medium outlet of the culture tank according to claim 1 through a contact portion with D0 and a pH sensor. (2) Constant temperature water circulating means between the circulation constant temperature tank and the culture tank jacket. (3) Gas mixture control and mixing means. (4) A means for ventilating the mixed gas to the oxygen permeable membrane. (5) Medium storage and medium supply means. (6) Means for extracting and storing used medium. (7) At least a horizontally rotatable ball type rotor, a liquid transfer pipe having an opening end close to the inner bottom surface of the rotor on the rotation axis of the rotor, and vertically movable blades, and these are housed in a pressure resistant chamber. Centrifugal means. (8) The centrifuge, the culture tank, the medium suspension storage means of the microcarrier, and the carrier-decomposing enzyme solution storage means are liquid transfer pipes, and the centrifuge and the gas mixture adjustment and mixing means are gas-transferred. Structure that connects with piping.