JPH0536026B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a serum-free or low protein concentration medium for culturing animal cells or cells derived from animal cells, and relates to a fat culture medium characterized by containing milk and/or milk components. be. Animal cells and cells derived from animal cells are known to produce effective substances such as antibodies, interferon, insulin, and enzymes, and it is possible to obtain these effective substances by culturing these cells. being carried out or attempted. Traditionally, in order to culture these cells, it is almost always necessary to add serum to the culture medium. Fetal bovine serum, calf serum, etc. are generally used as serum, and the amount added is usually about 10%. However, these serums are expensive;
There are drawbacks such as inconsistent quality and limited supply. Furthermore, since serum contains a wide variety of components such as proteins, it is difficult to separate and purify effective substances from culture. As a result of repeated research to solve these problems, the present inventors discovered that cells can be grown in a serum-free medium with a low protein concentration, and have completed the present invention. Milk is, for example, human milk or animal milk (cow, goat, sheep, horse, etc.), and can be used regardless of whether it is colostrum or mature milk. A solution fraction obtained by centrifuging milk (hereinafter referred to as centrifuged milk solution fraction) is obtained by centrifuging milk at, for example, 20,000 x g. Molecular weight fractionation can be carried out using, for example, Sephadex G
−200 (Pharmacia Fine Chemicals) or
The gel filtration method is performed using Cellulofine GC-700 (Seikagaku Corporation) or the like. Equilibration and elution solutions used at that time include, for example, phosphate buffered saline, 100mM Na-Hepes buffer (PH
7.4), water, etc. Lactoferrin, for example
Lo¨nnerdal et al.'s method (Lo¨nnerdal, B., Carlsson,
J., and Porath, J. (1977) FEBS Lett.75, 89−
92). The cell culture medium of the present invention is used by adding these materials to a basal medium (not containing serum) used for normal animal cell culture. In addition, these milk and/or milk components,
Royal jelly and/or components of royal jelly, 1 from the fraction of placental blood with a molecular weight of 50,000 to 80,000
It is also possible to use seeds or more by adding them to the basal medium. The components of royal jelly are obtained as follows. The fraction of royal jelly with a molecular weight of 200,000 or more and the fraction with a molecular weight of 50,000 to 80,000 can be obtained in the same manner as the molecular weight fraction of milk. Royal jelly protein having a molecular weight of about 58,000 (hereinafter abbreviated as royal jelly protein) is purified, for example, as follows. Royal jelly is diluted twice with phosphate buffered saline and then dialyzed against it.
After dialysis, it is centrifuged at 20,000 x g, and the resulting supernatant is subjected to molecular weight fractionation in the same manner as for milk to obtain a molecular weight fraction of 50,000 to 80,000. After dialysis of this fraction against 1mM Tris-HCl buffer (PH8.0), Blue Sepharose (Pharmacia Fine
Chemicals) to obtain a flow-through fraction, that is, royal jelly protein. In addition, for the fraction of human placental blood with a molecular weight of 50,000 to 80,000 (hereinafter referred to as human placental blood fraction), for example, blood naturally exuded from the placenta is centrifuged at 20,000 x g, and the supernatant is further collected. Molecular weight fractionation can be performed by the method described above. As the basal medium of the culture medium of the present invention, any medium used for culturing general animal cells can be used. For example, Dulbecco's
Modified Eagle medium (hereinafter abbreviated as D'MEM),
Basal media such as F-12, RPMI-1640, insulin (2-10 μg/ml),
Selenium ( ^10-11-10-8 M), transferrin (10-35 μg/ ^ml ), nucleic acid precursors (e.g. thymidine, hypoxanthine, etc., about ^10-6 M), serum albumin (50-400 μg/ml), etc. A medium supplemented with can also be used as a basal medium. The milk added to the culture medium of the present invention is 0.1-5% (final protein concentration 7-350 μg/ml), preferably 0.5-5%.
It is desirable to keep it in the range of 2%. 7-350 μg/ml for the milk centrifugation solution fraction, 1-100 μg/ml for the fraction with a molecular weight of 200,000 or more, and a molecular weight of 4.
1-150 μg/ml for the −90,000 fraction and 0.5-50 μg/ml for lactoferrin. 10-300μ for other royal jelly, royal jelly components and placental blood fractions
g/ml is desirable. Although the culture medium of the present invention can be used for culturing animal cells such as fibroblasts and lymphoblastoid cells, it is most preferably used for culturing animal cell-derived cells such as lymphocyte-derived cells. Using the medium of the present invention, it is possible to obtain cell proliferation almost comparable to that of a conventional medium supplemented with a large amount of serum. In addition, it is cheaper than conventional media, and especially when milk and/or its components are used, culture can be performed very cheaply. In addition, compared to the protein concentration in the medium when 10% serum is added, which is 3 mg/ml or more, the medium of the present invention has a low protein concentration of 0.4 mg/ml or less, so it is effective for producing effective substances. By using the invented culture medium, it becomes easy to separate and purify the effective substance. Furthermore, since it is possible to add molecular weight fractions to the culture medium of the present invention, it can be used depending on the molecular weight of the effective substance. For example, when culturing cells that produce antibodies (molecular weight of 150,000 or more), lactoferrin and/or a fraction of milk with a molecular weight of 40,000 to 90,000 and, if necessary, a fraction with a molecular weight of about 40,000 to 90,000 separated and purified from royal jelly, are used.
58,000 proteins, the molecular weight of royal jelly is 5
-80,000 fraction and placental blood fraction are added and used as necessary. Furthermore, when culturing human cells in a medium supplemented with human milk components such as human lactoferrin and/or human milk and, if necessary, human placental blood fraction, all components in the medium are human-derived. Therefore, it has the advantage that the effective substance produced in the medium can be administered to humans without being completely purified. The present invention will be explained below with reference to Examples. Example 1 ¹⁰⁵ cells of a peripheral lymphocyte cell line (Bri7) from a healthy person were treated with D'MEM/F-12 with the additives shown in Table 1.
(1:1) in 2 mm of medium. 5
Culture was carried out for 3 days under conditions of % CO ₂ and 37°C. The results obtained are shown in Table 1.

[Table] Four additives were added to D'MEM/F-12: human lactoferrin alone, human lactoferrin and royal jelly protein, human serum albumin and lactoferin, and human serum albumin and lactoferrin and royal jelly protein. No change was observed in the respective growth rates even after subculturing in the medium for 30 days or more.
This indicates that continuous subculture is possible. Example 2 Human-derived lymphocytes (NL-matsumoto) transformed with Epstein-Barr Virus are cultured in the same manner as in Example 1. The results obtained are shown in Table 2. Table 2 Media final concentration of additives Number of cells (μg/ml) (cells) D'MEM/F-12 0.9×10 ⁵ + human lactoferrin 10 7.4×10 ⁵ + human serum albumin 200 4.0×10 ⁵ + Human lactoferrin 10 7.8×10 ⁵ + Fetal bovine serum, 20% 6400 8.2×10 ⁵

[Claims]

1. A fraction obtained by extracting royal jelly with water or phosphate buffered saline and then dialysis, or a fraction of royal jelly proteins with a molecular weight of 200,000 or more, or a fraction with a molecular weight of 50,000 to 90,000, preferably a molecular weight
1. A cell culture medium containing royal jelly components and/or royal jelly selected from 58,000 fractions. 2 Royal jelly selected from the fraction obtained by extracting royal jelly with water or phosphate buffered saline and then dialysis, or the fraction of royal jelly proteins with a molecular weight of 200,000 or more, or the fraction with a molecular weight of 50,000 to 90,000, preferably the fraction with a molecular weight of 58,000. ingredients and/or
Or royal jelly, a solution fraction obtained by centrifuging milk, a milk protein fraction with a molecular weight of 200,000 or more or a fraction with a molecular weight of 40,000 to 90,000, or milk components consisting of lactoferrin, milk, placental blood with a molecular weight of 5 −
A cell culture medium characterized by containing an additional component selected from 80,000 fractions.