JPH06113828A - Additive composition for serum-free medium - Google Patents

Abstract

PURPOSE:To provide the subject additive composition for a serum-free medium, capable of exhibiting an excellent cell growth promotion activity, of efficiently culturing cells and of readily separating and purifying the produced useful materials while keeping the constant quality at low cost. CONSTITUTION:This additive composition for a serum-free cell culture medium contains royal jelly-derived proteins, especially the DIII fraction obtained by isolating through an ion-exchange column. This DIII fraction is added in an amount within the range of 0.1mug/ml to 4mg/ml to an eRDF medium as a serum- free medium containing amino acids, sugars and vitamins. If human monocytoid cell (U937) is cultured in this culture medium, an excellent cell growth promotion effect is exhibited.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an additive composition for a serum-free medium which exhibits a cell growth promoting effect and the like when added to a serum-free medium for culturing animal cells, for example.

[0002]

2. Description of the Related Art Conventionally, in the production of useful substances by animal cells, a medium supplemented with about 10% of animal serum has been used in order to improve productivity.

[0003]

However, since such a medium contains a high concentration of protein derived from serum, the purification method for taking out a useful substance from the obtained product solution becomes complicated. . Moreover, animal serum has a large difference in quality depending on the production lot, is not always stably supplied, and is expensive, which causes a problem of increasing the production cost of useful substances by animal cells. .

The present invention has been made in view of such conventional problems, and an object thereof is to exert an excellent cell culture activity such as a cell growth promoting activity and enable efficient cell culture. Another object of the present invention is to provide an additive composition for serum-free medium, which can easily separate and purify the produced useful substance, and can supply stable quality at low cost.

[0005]

To achieve the above object, the additive composition for serum-free medium for cell culture of the present invention is characterized by containing a protein derived from royal jelly.

Next, the present invention will be described in more detail. Royal jelly is produced by bees, and the queen bee ingests only this royal jelly for life, and produces a huge number of eggs. This is because royal jelly has a rich nutritional and diverse physiological action, which is said to be a complete food. That is, this physiological action is an anticancer action, an antitumor action, a wound healing promoting action, an antibacterial action, an antidiabetic action, a blood flow increasing action, an anti-atherogenic action, a growth promoting action and the like. This means that the physiologically active substance is present in the royal jelly. Focusing on these points, royal jelly is used as health foods, cosmetics, pharmaceuticals, and the like.

In animal cell culture, the best growth promoting effect can be obtained by adding the protein fraction in such royal jelly to various serum-free media. That is, various proteins can be efficiently cultured by separating and purifying proteins from royal jelly and adding the obtained proteins in the range of 0.1 μg / ml to 4 mg / ml with respect to the amount of medium.
And, it is easy to separate the useful substance from the product. Therefore, the cell culture can be effectively performed without adding the conventionally used proteinaceous growth-promoting factors such as insulin, fetal bovine serum, and protein components such as cytokines.

The protein in royal jelly, that is, the factor showing the growth promoting activity, can be easily separated and purified as follows. That is, the protein fraction obtained by a conventional method is separated and purified in a high yield by a method such as gel filtration, ion exchange chromatography, hydrophobic chromatography, electrophoresis, high performance liquid chromatography using reversed phase column chromatography and the like. The obtained protein is fractionated by, for example, ion exchange chromatography. Therefore, the obtained protein derived from royal jelly can be obtained as a single protein as compared with the conventional proteinaceous growth factors, and the quality difference between production lots is small, and the protein can be obtained at low cost.

The DI fraction, D, thus fractionated
Fractions II and DIII are obtained. Of these fractions
The DIII fraction has the highest cell growth promoting ability. This DIII
The fraction is a glycoprotein fraction having an isoelectric point of 4.4 to 5.4 and a molecular weight of 300,000 to 400,000, which is composed of about 6 subunits having a molecular weight (weight average molecular weight) of 40,000 to 60,000. This glycoprotein fraction was dialyzed and lyophilized to give a powder.
If stored at or below ℃, it will be stable for several years, and the activity of promoting animal cell growth will be retained.

[0010]

Embodiments Embodiments embodying the present invention will be described below with reference to FIGS. (Example 1, production method of glycoprotein having cell growth promoting action) 107 g of raw royal jelly was mixed with 50 mM phosphate buffer (pH 6.2), and low molecular weight compounds were removed by dialysis, that is, a semipermeable membrane to obtain a molecular weight. Over 10,000 protein fractions were obtained.
Centrifugation (20,000 G, 20 minutes) for this fraction
Was performed to obtain a supernatant. This supernatant was preliminarily equilibrated with the above-mentioned phosphate buffer solution using an ion exchange column (DEAE-TOYOPEARL 650
After elution of the non-adsorbed fraction, the adsorbed fraction was linearly increased to a sodium chloride concentration of 0 to 0.3 M for elution.

As a result, as shown in FIG. 1, DI to DII
I fraction was obtained. DI fraction is 2.24 g (protein recovery rate 3
1.4%), DII fraction 0.2g (protein recovery rate 2.8%),
The DIII fraction was 3.15 g (protein recovery rate 44.2%).
The elution conditions in the ion exchange column were set as follows.

Column size: 1.5 cm x 38 cm, flow rate: 43.8
ml / hr, buffer (start): 50 mM sodium phosphate buffer (pH 6.2) (elution): 50 mM sodium phosphate buffer (pH
6.2) NaCl at concentrations up to +0.3 mM In addition, fraction No. in Fig. 1 is 1 fraction
It is 5.9 ml per No. (Test Example 1, method for measuring cell growth promoting action) Cell culture was performed as follows. Human monocytic cells (U937) and human-human hybridomas (HB4C5) producing human lung cancer-specific antibodies were used as the cultured cells for examining the growth-promoting effect. A serum-free medium containing sugar and vitamins (eRDF medium, etc., see Japanese Patent Laid-Open No. 4-51891) with ITES (insulin, transferrin, ethanolamine, sodium selenite) is used, and U937 is required for the eRDF medium. 5% FBS (fetal bovine serum) according to
It was carried out by culturing at 37 ° C. in 5% CO ₂ using the medium to which was added.

The presence or absence of cell growth promoting activity and cell growth factor activity was judged by the degree of increase in the number of viable cells due to the addition of royal jelly glycoprotein. The number of viable cells can be measured by MT
T [3-4,5-Dimethylthiazol-2-yl) -2,5-Diphenyl Tetra
zolium Bromide] -assay. This MT
The T-assay method is the method of Scudiero et al. (Cancer Research
48, 4827, 1988) with some modifications.

That is, first, cells cultured in a 24-well plate were dispensed in 110 μl aliquots into a 96-well plate, and MT
20 μl of T reagent was added. After reacting for 4 to 5 hours, the mixture was centrifuged and the supernatant was removed.
It was carried out by adding isopropanol containing 04N hydrochloric acid and measuring the absorbance at 540 nm. (Test Example 2, U937 cell growth promoting action) The serum-free medium was prepared by adding the royal jelly-derived DI fraction, DII fraction and DIII fraction to the eRDF medium in the range of 0.1 μg / ml to 4 mg / ml. Obtained. U937 using this medium
The cells were cultured for 5 days. The result is shown in FIG. As shown in FIG. 2, the number of viable cells in the DIII fraction was the highest, and the cell proliferation activity was the best. In particular, DIII
A peak appeared when the amount of the fraction added was 2 mg / ml, and about 7 times as much growth-promoting activity as that of the control (Comparative Example in which only FBS was added to the eRDF medium as a medium) was observed.

Following the DIII fraction, the DII fraction showed a high cell proliferation activity. Addition amount of DI fraction is 0.1 μg / ml 〜 1
It showed cell proliferation activity in the range of mg / ml. Although not shown, cell growth factor activity was also expressed in comparison with the control.

The present invention is not limited to the above-mentioned embodiments, but may be embodied by changing the structure as follows, for example, without departing from the spirit of the invention. (A) The cell culture is performed by adding the DI fraction or DII fraction as a protein derived from royal jelly to a serum-free medium. (B) Applying the present invention to culturing plant cells.

[0017]

As described above in detail, according to the present invention,
Efficient cell culture can be performed with excellent cell growth promoting activity and other cell culture activities, product separation and purification can be easily performed, and stable quality products can be supplied at low cost. It has an excellent effect.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the fraction No. and the absorbance of royal jelly proteins in Examples of the present invention.

FIG. 2 is a graph showing the relationship between the amount of protein and the number of viable cells in the same example.

Claims (1)

[Claims] 1. An additive composition for serum-free medium for cell culture, which comprises a protein derived from royal jelly.