JPS59173078A - Medium for cell culture - Google Patents

Abstract

PURPOSE:To provide a medium free from blood serum or having low protein concentration and suitable for the cultivation of animal cell or a cell originated from animal cell, by using royal jelly or its component as an essential component. CONSTITUTION:The objective medium is prepared by adding royal jelly and/or the component of royal jelly to a serum-free base medium for the conventional cultivation of animal cell. The medium may contain further, one or more substances selected from milk and/or milk component and a placenta blood fraction having a molecular weight of 50,000-80,000.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a serum-free medium with a low protein concentration for culturing animal cells or cells derived from animal cells, and is characterized by containing royal jelly and/or components of royal jelly. This invention relates to a cell culture medium.

Animal cells and cells derived from animal cells are known to produce effective substances such as antibodies, interferon, insulin, and enzymes, and it is difficult to obtain these effective substances by culturing the cells. Attempts are being made. Traditionally, in order to culture these cells, it is often necessary to add serum to the culture medium. As the serum, fetal bovine serum, calf serum, etc. are generally used, and the amount added is usually about 10%.

However, these serums have drawbacks such as being expensive, having 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.

Royal jelly is obtained from bees, etc., and can be obtained from, for example, the Kukeen Bee Garden Beekeeping Association, an agricultural cooperative corporation.

Royal jelly has a high viscosity, so it can be prepared using, for example, phosphate buffered saline, 100 mM Na-Hepes buffer,
Subsequent processing is facilitated by using both fractions (hereinafter referred to as royal jelly extraction and dialysis fractions) whose viscosity has been reduced by extraction and dialysis with water or the like. The molecular weight fraction is, for example, 8 ephad
ex G-200 (PharmaciaFine C
chemical) or Celulofine
This is carried out by gel filtration using GC-700 (Seikagaku Corporation) or the like. The equilibration and elution solution used at that time is, for example, phosphate buffered saline, 100 mM
These include Na-Hepes buffer (pH 7.4), water, and the like. The protein in royal jelly with a molecular weight of approximately 58,000 (hereinafter abbreviated as royal jelly protein) is
For example, purify as follows. Royal jelly is diluted 2 times with phosphate buffered saline and then dialyzed against it. After dialysis, it is centrifuged at 20,000 x 7, and the entire supernatant obtained is subjected to molecular weight fractionation using the above method to obtain both fractions with molecular weights of 50,000 to 80,000. Both fractions were dialyzed against 1 mM Tris-tIcjt buffer (pI (8,0)), and then Blue 8epharose (Pharma) equilibrated with the same buffer was added.
Cia Fine Chemicals) and subjected to clear DS electrophoresis (Weber, K., and 0
sborn, M. (1959) J., Biol, C.
Hem, 244, 4406-4412) showed a single band, and the molecular weight was determined by the same electrophoresis, and it was found to be 58. It was OOO. The cell culture medium of the present invention is
These materials are used by adding them to a basal medium (not containing serum) used for normal animal cell culture.

Furthermore, one or more of these royal jelly and/or royal jelly components, milk and/or milk components, and placental blood with a molecular weight of 50,000 to 80,000 may be added to the basal medium. .

The milk components are obtained as follows.

Milk is centrifuged, for example, at 20,000×2 to obtain a solution fraction. Both obtained fractions are subjected to molecular weight fractionation in the same manner as in the case of royal jelly, to obtain both fractions with a molecular weight of 200,000 or more and a fraction with a molecular weight of 40,000 to 90,000. Lactoferrin is, for example, L”one
The method of Rdal et al.

B., Carlsson, J+, and Por.
ath, J. (1977) FEBS Letter
, 7E5, 89-92) Te111M Sufu:r.

In addition, for both human placental blood fractions 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 2, and the supernatant is can be obtained by further molecular weight fractionation using the method described above.

As the basal medium of the culture medium of the present invention, any medium can be used as long as it is used for culturing general animal cells. For example, Dulbecco's Modifi
Basal media such as ed Eagle medium (hereinafter abbreviated as D'MEM), F-12, RPMI-1640, S- In addition, incirin (2-10 pf/
ml), selenium (10-10M), transferrin (10-35p?/ml"), nucleic acid precursors (e.g. thymidine, hypoxanthine, etc., about 10M),
A medium supplemented with serum albumin (50-400 pf/ml) can also be used as the basal medium. The royal jelly added to the medium of the present invention is 0.003-0
．． 3% (final concentration of protein is 3-30011 f/m
l), 0.01-0.2% range of 90,000 and 10-2 for royal jelly protein
00 μf/ml is desirable. 0.5-350 μt/ml for other milk and milk components.

For both placental blood, it is desirable to be 50-300 μt/ml.

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 culture medium of the present invention, it is possible to obtain cell proliferation that is comparable to that of a conventional culture medium supplemented with a large amount of serum. Also, compared to the conventional method of adding 10% serum, the amount of royal jelly added is 0.0%.
It only requires as little as 1-0.2%, which has the advantage of being inexpensive and reducing crowding.8For example, when comparing the protein concentration in the culture medium, when 10% serum is added, it is more than 3 rq/ml. Compared to this, the medium of the present invention has a low protein concentration of 0.41 Ity/ml or less. Furthermore, since it is possible to add molecular weight fractions to the culture medium of the present invention, it is possible to use 1 gJ depending on the molecular weight of the effective substance. For example, when culturing cells that produce antibodies (molecular weight 150,000 or more), royal jelly protein and/or the 50,000 to 80,000 molecular weight fraction of royal jelly, lactoferrin, both milk fractions with a molecular weight of 40,000 to 90,000, and placental blood fractions are used. Add and use as necessary. As described above, the use of the culture medium of the present invention for the production of effective substances has the advantage that the effective substances can be easily separated and purified.

The present invention will be explained below with reference to Examples.

Example 1 Ten peripheral lymphocyte cell lines (Bri 7) from healthy individuals are implanted in 2 ml of a medium containing D'MEM/F-12 (1:1) and the additives shown in Table 1. 5%C
Culture is carried out for 3 days under the conditions of O2 and 37°C. The results obtained are shown in Table 1.

Table 1 Media Final concentration of additives Number of cells Cμf/
Tll> (units) + fetal bovine serum, 10% to 200
9.8×10′ The same applies to other examples.

Furthermore, even when cultured for more than 30 days in a medium containing MEM/F-12 and three additives: royal jelly protein alone, royal jelly protein and human lactoferrin, and human serum albumin and royal jelly protein and human lactoferrin, each No change was observed in the growth rate. This indicates that continuous subculture is possible.

Example 2 Human-derived lymphocyte cell line (NL-77) transformed with Epstein-Barr Virus
Example 1
Culture is carried out in the same manner as above. The results obtained are shown in Table 2.

Table 2 Final concentration of additives Number of cells (μg
/wl) (cells) -ter Example 3 Human-derived lymphocyte cell lines (HYON) are cultured in the same manner as in Example 1. The results obtained are shown in Table 3.

Table 3 Media Final concentration of additives Number of cells (μf/
Id> (units) *+Fetal bovine serum, 2% and horse serum, 6% 4.70
0 t9xlo' quality concentration.

Patent applicant: Morinaga & Co., Ltd. -lθ-

Claims (1)

[Scope of Claims] 1. A cell culture medium characterized by containing royal jelly and/or royal jelly components. 2. The components of royal jelly are extracted and dialyzed from royal jelly with water or phosphate buffered saline, molecular weight of 1.2 million or more, molecular weight of 50,000 to 80,000, or molecular weight of approximately 58. The cell culture medium according to claim 1, which is an OOO protein. 3 Royal jelly and/or royal jelly components, milk and/or milk components, molecular weight of placenta surface 5-
A cell culture medium characterized by containing one or more types selected from among 80,000 types. 4. Claim 3, wherein the milk component is a solution fraction obtained by centrifuging milk, a fraction with a molecular weight of 200,000 or more, a fraction with a molecular weight of 40,000 to 90,000, or lactoferrin.
The cell culture medium described in Section 1.