JPH0576344A - Floating cell culture method and culture device - Google Patents

Abstract

(57) [Summary] [Structure] The upper part of the culture chamber 6 is sealed by a lid 7, and a sealing cap 21 at the central portion allows a supernatant extraction pipe 11 penetrating the lid 7, an oxygen-containing gas supply pipe 14, an oxygen-containing pipe. The gas exhaust pipe 16, the D0 electrode 17, and the pH electrode 19 are fixed and located in the culture tank 1. Supernatant extraction pipe 1
One end of a stirring blade 22 is connected to the lower end portion of the blade 1, and the tip of the blade 22 is freely moved up and down in the vertical direction with the lower end of the pipe 11 as a fulcrum so that the blade can be opened and closed. [Effect] By using the rotor as a culture tank, the volumetric efficiency can be greatly improved compared to the conventional large scale, and thus perfusion culture can be performed on a small scale.

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 buoyant organism cells, and more particularly to a perfusion culture method and a perfusion culture apparatus suitable for small-scale culturing of buoyant animal cells.

[0002]

2. Description of the Related Art High-density perfusion culture of free-floating cells, especially free-flowing animal cells, is becoming a key technology for producing proteinaceous physiologically active substances. Among them, the centrifugal type perfusion culture method is considered to be promising because it has high durability, can endure long-term culture, and can achieve high perfusion rate. Known techniques include, for example, Japanese Patent Application Nos. 63-143466 and 63-282544. In these conventional methods and culture devices, the culture solution in the culture tank is extracted to the outside of the tank, and the medium is replaced by a centrifugal separator devised for animal cells.
In addition, in order to prevent the cells from coming into contact with the high-speed rotating surface and being damaged during centrifugation, it is necessary to pull the stirring blade out of the rotor together with the shaft tube or rotate it in synchronization with the rotor, so a robust and complicated mechanism There was no choice but to become.

However, perfusion culture on a small scale of about 1 liter is also required for seed culture before main culture and culture at the stage of product search. However, it is extremely difficult to scale down as it is by the above-mentioned known technique, and a culture device that can withstand a long-term perfusion culture at a scale of 1 liter or less is not known.

[0004]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method and system that enables perfusion culture for a long period of several months even on a small scale of 1 liter or less.
For that purpose, it is necessary to devise a process and a structure that make the function of the device compact without detracting from it.

[0005]

Means for Solving the Problems The inventors of the present invention have earnestly studied the principle required for compactification, and as a result, arrived at the present invention by changing the way of thinking that the rotor of a centrifuge is a culture tank.

The first feature of the present invention is a process including the following steps.

That is, (1) the medium and seed cells are introduced into a horizontally rotatable culture tank, and (2) the oxygen supply means fixed on the central axis of rotation and the blade base are fixed, and the base to the blade tip is fixed. With the blades moving above and below the liquid surface positioned below the liquid surface, the culture tank is rotated at a low rotation speed at which cells do not substantially settle, thereby stirring the liquid and dissolving oxygen for a predetermined period of time. 3) Pulling the rotor surface to the liquid surface, rotating the culture tank at a rotation speed sufficient for the cells to be centrifugally sedimented, rotating until the cells are deposited on the inner surface of the culture tank, and then stopping the rotation,
(4) The supernatant generated by centrifugation was extracted to the outside of the system via a liquid transfer pipe having an opening close to the bottom of the culture tank and fixedly arranged on the rotation axis, and (5) provided at the blade tip. While rotating at a low speed in the state, the culture medium is radiated from above the inner wall of the culture tank to disperse the deposited cells in the culture medium, and (6) the process returns to the step (2), and thereafter (2) to (6). Repeat the cycle.

Further, FIG. 1 schematically shows the present process.
Will be described in more detail. In the culture tank 1 having the function of a rotor, a stirring blade 2 having a base portion and capable of moving the blade tip up and down is arranged at the lower end of the supernatant withdrawing pipe 10 at the time of culture.
The culture solution 4 is mixed by rotating the culture tank 1 in the state of opening. During this time, an oxygen-containing gas is introduced from the oxygen supply pipe 8 into the oxygen-permeable hollow fiber membrane 12 fixed to the pipe 10 to dissolve oxygen in the culture solution 4. When the medium needs to be exchanged, the blade tip of the stirring blade 2 is raised and the culture tank 1 is rotated at a rotation speed sufficient for centrifugation to settle the cells 5 on the inner wall of the tank. Then, the supernatant 6 is extracted from the pipe 10. Next, the medium is radiated from the medium supply pipe 11 to suspend and disperse the cells attached to the inner wall of the tank in the medium. At this time, the culture tank 1 rotates at a low speed with the wings 2 open.

The cells applicable to the present invention are not particularly limited as long as they are buoyant, but are preferably buoyant animal cells such as various hybridomas, cancer cells, lymphocyte cells and the like.

[0010]

By using the rotor of the centrifuge as a culture tank, perfusion culture becomes possible, and the volumetric efficiency can be greatly improved.

[0011]

EXAMPLES Next, examples of the present invention will be shown and described in more detail.

Example 1 FIG. 2 shows a cross section of the core portion of the culture apparatus according to the present invention. The culture tank 1 which can be freely rotated horizontally has a magnet 2 for transmitting a rotational force at its lower portion, and is supported by a bearing 4 and a support arm 5 at the top and bottom and is housed in a pressure-resistant culture chamber 6. A motor 3 provided with a magnet 2 may be arranged outside the bottom of the culture chamber 6 to rotate the culture tank. The upper part of the culture chamber 6 is sealed with a lid 7, and a supernatant removal pipe 11 penetrating the lid 7, an oxygen-containing gas supply pipe 14, an oxygen-containing gas exhaust pipe 16, a D0 electrode 17, and a sealing cap 21 at the central portion. The pH electrode 19 is fixed and located in the culture tank 1. Supernatant extraction pipe 11
One end of the stirring blade 22 is connected to the lower end of the blade, and the tip of the blade 22 is freely moved up and down in the vertical direction with the lower end of the pipe 11 as a fulcrum to open and close the blade.

A cable line is laid slightly toward the tip of the wing from the fulcrum of the wing, and a wing 2 is formed by a solenoid 23 and an operation transmission line 24.
Open and close 2. In addition, the oxygen permeable hollow system 6a is fixed to the supernatant extraction pipe 11 and the oxygen-containing gas 13 is introduced to supply oxygen into the culture solution in the culture tank. During the culture, the culture tank 1 is rotated at a low speed with the blades 22 open to stir the inside of the culture tank, and D0 and pH are detected by the respective electrodes, and it is possible to control within each set range. During centrifugation,
Raise the blades 22 upward so that they are separated from the liquid surface, and rotate the culture tank at 800 to 2000 rpm (1000 G or less). When the cells are suspended in the medium, they are emitted from the medium supply pipe 27 to the inner surface of the tank. Reference numeral 25 is a drain discharge pipe.

Example 2 FIG. 3 shows an example of a culture system in a single culture tank.

The medium 18 is supplied to the culture tank 1 by the horn 1.
2. The pump 13 extracts the supernatant 16.
The oxygen-containing gas is supplied to the oxygen-permeable hollow fiber membrane 6 through the pipe 7. The gas supply, the pump operation, and the opening / closing of the stirring blade 5 are performed by the D0 / pH controller 15 and the process sequencer 14.

9 is a supernatant, 10 is a pipe for extracting the supernatant, 11
Is a medium, 12 is a medium supply pipe, 13 is an oxygen-containing gas supply pipe, 14 is an oxygen-containing gas supply pipe, 15 is an oxygen-containing gas waste gas, 16 is an oxygen-containing gas waste gas pipe, 17 is a pH electrode,
Reference numeral 18 is a pH electrode signal wiring, 19 is a D0 electrode, 20 is a motor wiring, and 21 is a culture chamber.

<Embodiment 3> FIG. 4 shows an example of a culture system having a plurality of tanks. The supernatant extraction pipe 11, the medium supply pipe 10, and the gas supply pipe 7 can be assembled by operating the opening / closing valve for each tank.

[0018]

According to the present invention, since the rotor culture tank is used, the volumetric efficiency can be greatly improved as compared with the conventional large scale, so that the perfusion culture can be performed on the small scale.

[Brief description of drawings]

FIG. 1 is an explanatory diagram of a process principle of the present invention.

FIG. 2 is a sectional view of a main part of the culture device of the present invention.

FIG. 3 is a flowchart of a culture system in which the culture tank of the present invention is a single tank.

FIG. 4 is a flowchart of a culture system of the present invention when there are a plurality of culture tanks.

[Explanation of symbols]

1 ... Culture tank, 2 ... Magnet, 3 ... Motor, 4 ... Bearing, 5 ... Support arm, 6 ... Culture chamber, 22 ... Stirring blade.

Claims (2)

[Claims] 1. A method for culturing floating cells, which comprises the following steps. (1) The medium and seed cells are introduced into a horizontally rotatable culture tank, (2) the oxygen supply means fixed on the central axis of rotation, and the blade base are fixed, and the liquid level is raised and lowered from the base to the blade tip. (3) The stirring of the liquid and the dissolution of oxygen are performed for a predetermined period of time by rotating the culture tank at a low rotation speed at which cells are not settled by centrifugal sedimentation with the impeller positioned below the liquid surface. The rotor is pulled up to the liquid surface, and the culture tank is rotated at a rotation speed sufficient for centrifugal sedimentation of the cells to rotate the cells until they are deposited on the side surface in the culture tank, and then the rotation is stopped, (4) The supernatant produced by centrifugation is withdrawn to the outside of the system via a liquid transfer pipe having an opening close to the bottom of the culture tank and fixedly arranged on the rotation axis, and (5) a state in which the blade tip is lowered. While rotating at low speed, the culture medium is radiated from above the inner wall of the culture tank and deposited. The cells are dispersed in the medium, the process returns to (6) and (2), and then the cycles (2) to (6) are repeated. 2. A culturing device for floating cells, which has the following structure. (1) A horizontally rotatable ball-type culture tank has an opening near the center bottom surface of the culture tank on the axis of rotation and a liquid transfer pipe on the axis of rotation. (2) An oxygen supply means is provided in the liquid transfer pipe, and (3) a base is fixed on the upper and lower sides of the liquid transfer pipe, and a stirring blade is provided which allows the end part to be moved up and down with the base as a base point.