JPH0728729B2 - Method for culturing animal cells - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application The present invention relates to a method for culturing animal cells. More specifically, it relates to a method for culturing animal cells producing a useful substance at high density.

(B) Conventional Technology Large-scale cell mass culture is essential for the production of viruses, vaccines, antiviral agents such as interferons, or biopharmaceuticals such as hormones. In particular, in recent years, the production of monoclonal antibodies targeting specific proteins and the like is based on large-scale culture of hybridomas with antibody-producing cells and myeloma, and the solution of the technique is an industrially important theme.

Conventionally, some proposals have been made as a method for mass culturing cells and a device therefor. However, in these methods, particularly in the culture in a serum-free medium, they are not grown to a sufficiently high density industrially.

(C) Purpose of the Invention Therefore, the present inventors have aimed to grow a large amount and a high density of a method which was conventionally able to grow only about 10 ^{6 to} 5 × 10 ⁶ cells / ml in suspension culture. As a result of conducting research, the present invention has been reached.

(D) Structure and Effect of the Invention That is, the present invention provides a method in which animal cells are suspended in a culture tank, a new culture solution is supplied to the culture tank, and an old culture solution is discharged to the outside of the culture tank. In the method of discharging and culturing at high density, the culturing is performed using a serum-free medium and in the presence of polyvinylpyrrolidone in the suspension culture, which is a method for culturing animal cells.

According to the present invention, animal cells can be effectively proliferated and can be grown at high density.

Hereinafter, the present invention will be described in more detail.

The cell culturing method of the present invention is applied to a method of culturing in a suspension state using a serum-free medium, and the suspension state means that the cells themselves are suspended in an aqueous medium or the cells are microcarriers ( It refers to various suspension cultures in which cells are carried on microcarriers and suspended and cells are grown in microcapsules. In particular, the present invention is advantageously used in a method of culturing cells themselves while suspending them.

In the culturing method of the present invention, the animal cells to be cultured may be those capable of growing in a suspended state, and not only natural animal cells but also artificially or genetically modified cells such as hybridomas, Good.

The cells may be lymphocyte-derived cells that produce lymphokines such as IL-2-, or diploid cells that produce useful physiologically active substances such as interferon (IFN). Further, it may be cells producing various monoclonal antibodies, and the present invention is particularly suitable for the purpose of obtaining a high concentration of the monoclonal antibody in the culture of hybridomas producing such monoclonal antibody.

The cell culture in the present invention is performed by the perfusion method. Here, the perfusion method is a method of generally culturing while supplying a new culture solution into a culture tank and discharging an old culture solution containing a growth-inhibiting substance to the outside of the culture tank. One of the important things in culturing using this method is that the living cells in the suspension liquid and the old culture liquid are efficiently separated, and the old culture liquid is taken out of the culture tank, To maintain the cell growth environment under optimum conditions. In addition, the old culture solution separated from the suspension solution and taken out of the culture layer is removed from the growth inhibitor contained in the old culture solution by a membrane separation method or a separation method by adsorption. After fractionating the useful substance produced by the above, it can be reused as a new culture medium by newly adding additional components necessary for culture.

In the suspension state cell culture of the present invention, in the culture tank, the cells to be cultured are cultured in a state of being suspended in the culture medium. The culture solution is a so-called serum-free medium, and in an aqueous medium consisting essentially of water, various inorganic salts, vitamins, scavenging enzymes, grapes, amino acids, antibiotics and other additional components usually used for cell culture, Growth factors commonly used in serum-free medium and polyvinylpyrrolidone are added. Further, additives such as serum albumin and polyglycol can be added to the culture medium together with polyvinylpyrrolidone.

Here, polyvinylpyrrolidone is represented by the following formula (I) Polyvinylpyrrolidone is a compound comprising 100 to 6,000 of these repeating units. Polyvirpyrrolidone used in the present invention is soluble in water, its molecular weight is 10,000 ~ 700,000,
It is preferably 40,000 to 400,000. The amount added to the suspension liquid is 0.1 to 10 g /, preferably 0.5 to 5 g /
It is advantageous to add.

In the culture method of the present invention, oxygen or an oxygen-containing gas may be directly supplied into the suspension liquid as a method of supplying oxygen in order to keep the oxygen concentration in the suspension liquid constant. Although other supply means may be used, the present invention is particularly effective for directly supplying oxygen or oxygen-containing gas into the suspension liquid. As another supply means, for example, there is a method using an oxygen carrier.

As the oxygen carrier, a liquid compound capable of dissolving oxygen without being substantially mixed with water is used, and examples thereof include various fluorocarbons used as a material for artificial blood. When such a fluorocarbon is used as the oxygen supply means, the fluorocarbon in which oxygen is dissolved may be added in the form of a droplet or a thin film from above in the suspension liquid.

Thus, according to the method of the present invention, in the suspension culture in the perfusion system, the viable cell rate is increased to 10 ⁶ cells / ml or more, and 5 × 10 ⁶ cells / ml under the preferable conditions.
It is possible to culture at a high density of more than ml.

Hereinafter, the present invention will be described in detail with reference to examples.

Example (1) Culture device The culture system shown in the attached FIG. 1 was used. As shown in the figure, the culture tank is provided with a settling zone on the outer wall, which is partitioned by a partition wall inside, and has a culture solution discharge port at the upper part thereof, and the net culture volume is about 120 ml.

(2) Culture medium As a basal medium, RPMI1640 medium, Ham-F12 medium and Dulbecco's modified Eagle medium were mixed at a ratio of 2: 1: 1 to which amino acids, glucose and the like were further enhanced (hereinafter, e
-RDF), insulin as a growth factor,
Transferrin, ethanolamine, selenious acid (IT
ES) was added. The amount of insulin added was 9 μg / ml, transferrin was 10 μg / ml, ethanolamine was 10 μM, and selenious acid was 20 nM.

(3) Culturing method and results A culture medium a having a net culture volume of about 120 ml was fed to the aforementioned autoclave-sterilized culture tank, and mouse x human hybridoma 41 having mouse myeloma cell P3U1 strain as a parent strain was fed into the culture medium a. The 2-1 strain was seeded at 4.8 × 10 ⁵ cells / ml. This cell is an IgG-producing strain. Oxygen gas (so that dissolved oxygen is 3 ppm) was automatically controlled and fed into the culture tank through a nozzle B. The culture solution in the culture tank was kept at 37 ° C. A marine-type stirring blade is installed in the culture tank, and the stirring speed is 60 rp.
It was m.

Batch culture was performed for 1 day after seeding, and then perfusion was started. The perfusion scale is expressed as the replacement rate of the net culture volume per day, and is also shown in the experimental results.

The above culture operation was continued for 13 days. The results are shown in Table 1.

Comparative Example The culture device used was the same as that used in the example. The culture was performed in the same manner as in Example except that polyvinylpyrrolidone was not included. The culture was continued for 17 days. The results are shown in Table 2.

[Brief description of drawings]

The attached drawings show an example of a schematic view of steps for carrying out the culture method of the present invention.

Claims (1)

[Claims] 1. A method of culturing animal cells in a suspension tank in a suspended state, supplying a new culture solution into the culture tank, discharging the old culture solution outside the culture tank, and culturing at a high density. 2. A method for culturing animal cells, wherein the culturing is carried out using a serum-free medium and in the presence of polyvinylpyrrolidone in the suspension culture solution.