JPH037578A - Method and apparatus for culturing cell - Google Patents

Abstract

PURPOSE:To enable culture without foaming a culture medium by feeding oxygen through oxygen-permeable membranes of a hollow yarn bundle, arranged in a culture vessel and having free ends flowable with stirring of a liquid into a culture solution. CONSTITUTION:A bundle 7 prepared by collecting 10-800 hollow yarns 3 having 0.5-5mm outside diameter with binding parts 4 so as to provide free ends flowable with stirring of a culture solution is installed in a culture vessel 1 equipped with rotating blades 2. The culture solution in the vessel 1 is then stirred with the rotating blades 2 and oxygen is simultaneously fed from an outlet and inlet 5 and 6 into the hollow yarn bundle 7 through oxygen-permeable membranes, forming the hollow yarns 3 and having 0.05-0.5mum pore diameter into the culture solution.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a culture method and apparatus that improve the oxygen supply method for cell culture such as animal cells, particularly for mass culture.

(Prior Art) Generally, in the cultivation of microorganisms, the oxygen (air) necessary for the growth of microorganisms is supplied by a method called sparging, in which oxygen or air is directly blown into the liquid in a culture tank. Liquids such as serum in the culture medium in animal cell culture are extremely susceptible to foaming due to oxygen and air introduced into the culture (often causing problems in culture. Animal cells are also sensitive to physical forces). Very weak (cell damage occurs due to sparging).

For these reasons, conventionally, as shown in Figure 4, oxygen (air) is supplied only to the gas phase of the culture tank, and oxygen (air) is only supplied by the diffusion of oxygen (air) from the gas phase to the liquid phase. A method of supplying air) is known. With this method and device,
Although small-capacity culture tanks have sufficient oxygen supply capacity to some extent, large-capacity culture tanks such as those on an industrial scale,
When a large amount of culture is performed, the ratio of the air-liquid interface to the liquid volume becomes small, and the oxygen supply capacity becomes low and insufficient.

In order to improve the disadvantage described in L, a method has been reported in which an oxygen permeable membrane is coiled and placed in a culture tank to supply oxygen through the membrane without foaming.

(For example, M, Toyo, D, 1.C, Wang,
,Advan.

(Old Otech, 1.141H981)) This method has an oxygen supply capacity of 1 minute, and can be used for larger culture tanks.
iJ Noh.

(Problems to be Solved by the Invention) Methods for supplying oxygen through oxygen-permeable membranes have sufficient oxygen supply capacity for large-capacity culture and do not cause foaming, but these oxygen-permeable membranes The membrane is fixed in the tank, and the fixed membrane becomes an obstacle to the agitation and flow of the liquid necessary for cell culture, causing local turbulence in the liquid flow and causing abnormal shearing force in some parts. , damaging to cultures, especially animal cells. Therefore, it is an object of the present invention to provide a culture tank that minimizes abnormal turbulence in the liquid flow in the culture tank, has sufficient oxygen supply capacity, and has almost no foaming in the liquid.

(Means for Solving the Problems) That is, the present invention provides a method for culturing cells, etc. in a culture tank, in which the east of the hollow fiber has a free end that can flow in response to liquid stirring in the culture tank. The oxygen (
A method for culturing cells, etc., characterized by supplying oxygen (air) into the culture solution through an air) permeable membrane, and an apparatus for culturing cells, etc. in a culture tank, in which:
An apparatus for cultivating cells, etc., characterized in that a hollow fiber bundle for supplying oxygen (air) is arranged, and the hollow fiber bundle has a free end where flow is not substantially restricted by liquid stirring. It is.

Hereinafter, the present invention will be explained with reference to the drawings. FIG. 1 and FIG. 2 show an overview of an example of the present invention. 1 in the diagram
is a culture tank, 2 is a rotary blade for stirring the liquid, 3 is a hollow fiber bundle for oxygen supply, and 5 and 6 are ports for oxygen (air), respectively.

The hollow fiber bundle 3 is made up of a plurality of hollow fibers held together by a bundling part 4. In this example (Fig. 3), the bundling part is divided into two parts, and the fibers are fixed at two places in the culture tank. Although it has been
It is fixed with a single bundle part and oxygen is introduced into and out of the single bundle part 11.
may be provided and fixed in the tank by one fixed part. In either case, as the liquid (in the tank) flows, the end (free end) of the hollow fiber bundle, which is the part furthest from any of the bundled parts, can move substantially freely. It must be placed in the "Substantially freely movable" means that the free end moves 3 times, preferably 5 times or more, relative to the overall diameter of the hollow fiber bundle from a stationary state with no liquid flow. , more preferably 10 times or more of the twist, without suffering abnormal deformation such as breakage of the hollow fibers forming the hollow fiber bundle.

In the culture tank 1, the culture solution in the tank is uniformly stirred by rotary blades 2, and the oxygen (air) necessary for the cells is sent to the hollow fiber bundle from the inlets and outlets 5 and 6, forming hollow fibers. It is supplied to the culture solution in the tank by diffusion etc. through the oxygen permeable membrane. FIG. 3 is a schematic diagram showing an example of a bundled state of the hollow fiber bundle.

The hollow fiber bundle is made by binding a large number of hollow fibers 7 at a bundling part, and the hollow fibers are preferably made of a material with high oxygen permeability, such as silicone, but are not limited to it, and polypropylene, Teflon A. May be used.

The walls of these hollow fibers are formed porous, and if the pore diameter of the porous material is too large, foaming will occur, and if the pore diameter is too small, the oxygen permeability will be reduced. The preferred range of pore diameter is 0.05-0.5-
It is.

There are no particular limitations on the number of hollow fibers per τ1 method or the number of bundles to be installed, but if the diameter of the hollow fibers or wood is too small, they will be easily damaged during binding, and the outside diameter will be 0. ．． q~
Preferably, it is in the range of 5.

-・The number of hollow fibers on the east side is 10 to 10, depending on the liquid flow resistance etc.
800 pieces is preferable.

(Example) A hollow fiber membrane module made of silicon as shown in FIG. 3 was installed in a culture tank as shown in FIG. 1.

The hollow fiber membrane has an outer diameter of 1. In -, the number of membranes per module is 400 in the bundling part with an outer diameter of 50 m. Film length is 150
0, and was placed in a U-shape in a culture tank without fixing the bottom end. A 500C1 culture medium was placed in the culture tank, and oxygen was supplied into the culture medium. Air inlet has 20 Q,/mi
n air was flowed. K which shows the ability to supply oxygen into the tank in one dot.1. It was evaluated as +a.

Table 1 shows the relationship between K and the number of stirrings.

Unlike sparging, oxygen can be supplied into the culture medium without causing foaming of the medium, making the culture more efficient. Moreover, since the hollow system bundle is not fixed at the lower end of the liquid phase, the resistance to 1 m movement due to stirring is small, and liquid turbulence can be reduced, so that the culture efficiency can be increased to 1id-1- without cell damage.

[Brief explanation of drawings]

Fig. 1 shows a front view of the culture tank, and Fig. 2 shows a sectional view of the culture tank. FIG. 3 is a conceptual diagram of a membrane module (hollow system bundle). FIG. 4 shows a conventional oxygen supply method. (37°C) These values were sufficient for culturing animal cells, and no foaming was observed during oxygen supply. Moreover, the liquid flow in the liquid phase was smooth, and no local disturbances that could cause cell damage were observed. (Effect of the invention)

Claims (2)

[Claims] (1) In a method of culturing cells in a culture tank, a bundle of hollow fibers is arranged in the culture tank so as to have a free end that can flow in response to liquid stirring, and oxygen (air) permeation through the hollow fibers is performed. A method for culturing cells characterized by supplying oxygen (air) into the culture medium through a membrane. (2) In an apparatus for culturing cells in a culture tank, a hollow fiber bundle for supplying oxygen (air) is arranged in the culture tank, and the hollow fiber bundle has a substantial flow resistance against liquid stirring. 1. A cell culturing device characterized by having a free end that is not physically restrained.