CS260806B1 - Supplement of hybridoma and myeloma cell culture media - Google Patents

Abstract

The supplement for hybridoma and myeloma cell culture media is composed of at least one water-soluble iron compound. The supplement may further contain 2 x 102 mg of trace biogenic element compounds, 1 x 10”1 to 5 x 103 mg of ascorbic acid, 1 x 10-5 to 5 x 10”* mg of steroid substances and 1 x 10“2 to 7.5 x 102 mg of amines per 1 mmol of water-soluble iron compounds. This supplement is a suitable substitute for protein supplements such as blood serum or pure proteins

Description

The invention relates to the supplementation of culture media for hybridomas and myeloma cells.

So far, hybridomas and myeloma cells have been cultured in vitro on media which, in addition to essential components such as inorganic salts, monosaccharides, amino acids, vitamins, antibiotics, pH-buffering agents and pH-indicator, must contain proteins. It is known that hybridomas and myeloma cells cannot multiply in a culture medium without protein supplements. Therefore, up to 10% by volume of blood serum, such as bovine, horse or fetal calf serum, is added to the base medium. Blood serum introduces unknown substances into the culture medium, some of which promote the growth of hybrid and myeloma cells, others may inhibit growth. Blood sera must therefore be tested and selected before use, thus increasing the cost of producing culture media. Therefore, blood serum has been replaced in a number of culture medium recipes with a mixture of pure proteins, for example transferrin, serum albumin, insulin and possibly other substances such as unsaturated fatty acids, amines and low molecular weight hormones.

Proteins added to the culture medium must be purified to a high degree in order to be free from toxic impurities and therefore their cost is relatively high.

Another disadvantage of the culture media containing the protein supplement is the difficult isolation of the protein products secreted by the hybridomas or myeloma cells.

The deficiencies of protein supplements are overcome by a supplement according to the invention, which consists of at least one water-soluble iron compound, for example ferrous sulfate, ferric citrate, ferric chloride or potassium ferricyanide.

-4 2

The supplement may further comprise 2 x 10 to 1 x 10 mg-1 in 3 compounds of trace biogenic elements, 1 x 10 to 5 x 10 mg ascorbic acid, 1 χ'ΙΟ to 5 x 10 ¹ ng steroid and 1 x 10 to 7 5 x 10 mg of amines per 1 mmol of water-soluble iron compounds.

- 2 260 806

The supplement is added to the culture medium in an amount corresponding to a final concentration of 2 x 10 to 10 moles of water-soluble iron compound per 1 liter of culture medium.

It has been found that the only indispensable component of protein supplements is transferrin (Smith, J., Franek F., Methods in Enzymology 12, 277, 1986). Transferrin acts as an iron transporter from the culture medium to the cell. Its function can be replaced by the presence of a water-soluble iron compound of higher concentration.

Many authors (such as Phillips P.D., Cristofalo V.J.,

Exp. Cell Res., 134: 297,1981; Perez-Infante V., Mather J.P., Exp. Cell Res., 142: 325,1982; Taetle R., Rhyner K., Castagnola J., To D., Mendelsohn J., J. Clin. Invest., 75: 1061 (1985)) used water-soluble compounds as replacements for transferrin, with 6 lesions at concentrations of 1 x 10 to 20 x 10 mol / l. However, these concentrations are insufficient for hybridomas and myeloma cells.

Iron compounds at concentrations greater than 2 x 10 mol / L have been found to allow hybridomas and myeloma cells to grow and are not toxic to hybridomas and myeloma cells up to a concentration of 1 x 10 mol / L.

The advantage of replacing protein supplements with a supplement of the invention is a substantial reduction in the cost of the culture medium and easy isolation of protein products secreted by hybridomas or myeloma cells, since they are the only protein found after cultivation in the culture medium.

The use of the water-soluble iron compounds of the invention is illustrated by the following examples.

Example 1

The culture medium contained both the basic components of RPMI 1640 in which L-glutamine (300 µg / ml), N-2-hydroxyethylpiperazine-N * -2-ethanesulfonic acid (1.5 x 10 mol / l), penicillin (100 µg / ml) were dissolved. units / ml), streptomycin (100 µg / ml) and gentamycin, _ν 260 806 (40 pg / ml).

As a supplement, the culture medium contained 5 χ · ΐδ ^ mol / 1 ** 6 ferric citrate, 1 x 10 mol / 1 zinc sulfate, 2.5 "(9" 10 x 10 mol / 1 ammonium vanadate, 2.5 x 10 mol / 1 chloride

-7 stannous, 2 x 10 mol / l sodium silicate, 4 x 10 sodium selenite, 2.5 x 10 ^-10 nickel sulphate, 5 x 10 ¹⁰ mol / l '10-8 manganese chloride, 5 x 10 ammonium molybdate, 1 x 10 mol / l copper sulfate, 1 x 10 mol / l cobalt chloride, "8" 9 x 10 cadmium sulfate, 5 x 10 mol / l hydrocortisone, "5 -5 x 10 mol / l ascorbic acid and 2 x 10 mol / L ethanolamine.

PLV-01 and PGG-05 hybridomas were cultured in this medium.

After 4 days of culture, the number of cells increased from the original number of 60 x 10 6 cells per ml to 819 x 10 6 cells / ml for PLV-013 hybridoma and to 675 x 10 cells / ml for PGG-05 hybridoma.

Concentrations of monoclonal antibodies provided with enzymoimunologického assay based on the determination of peroxidase activity and expressed as relative absorbance units reached ^4G _O ^e 0.298 for hybridoma PLV-01 values ^{Α 49Ο β} ®2 l'P ^ro hybridoma PGG-05th

Example 2

The culture medium contained the basic ingredients as in Example 1. For comparison, a supplement containing 5 mg / l transferrin and 10 mg / l insulin was added.

PLV-01 and PGG-05 hybridomas were cultured in this medium.

After days of culture, the number of cells increased from the original x10® cell / ml to 706 x 10® cells and the ^Α 49θ * 0.310 for PLV-01 hybridoma. For the PGG-05 hybridoma, the number of cells increased to 731 x 10 and the ^A 490 ^S value was 1.26.

Example 3 260 808

PLV-O1 cells were inoculated at 10 x 10 ² cells into 0.1 ml of culture medium containing the basic ingredients as in Example 1 and containing 5 x ^{10 -5} mol / L ferrous sulfate as a supplement. After 3 days of culture * 3, cells were expanded to 26 x 10.

Claims (2)

object of the invention Supplement to culture media of hybridomas and myeloma cells, characterized in that it comprises at least one water-soluble iron compound, preferably ferrous sulfate, ferric citrate, ferric chloride or potassium fericyanide, 2. The supplement of claim 1 comprising: 2 x 10 to 1 x 10 mg of the compounds of trace elements biogenic 1 x IO ^1-5 x 10 ³ mg ascorbic acid, 1 x 10-5 x io ^{- 1} mg of steroid compounds, and 1 x '10 to 7.5 10 ^ χ mg of amines per mmol of water-soluble iron compounds.