ALTEX :: Alternatives to Animal Experiments

2001, VOLUME 1

Replacement of Fetal Calf Serum by an Egg Yolk Factor with CCK/Gastrin-like Immunoreactivity in Cell Culture

Mirko Sasse¹, Thomas Lengwinat³, Peter Henklein², Andreas Hlinak⁴ and Rüdiger Schade¹

¹Institute of Pharmacology and Toxicology and ²Institute of Biochemistry, Medical Faculty (Charité), Humboldt-University, D-Berlin; ³Institute of Zoo and Wildlife Biology, D-Berlin; ⁴State Veterinary and Food Investigation Centre, D-Frankfurt/Oder

The in vitro culture of different cell types is an important scientific tool and becomes increasingly accepted as a real alternative to animal experiments. Foetal calve serum (FCS) is a common supplement used in many cell culture mediums and provides cells with many growth factors and cytokines necessary for a successful culture. In view of the animal protection issues surrounding the production of FCS, an alternative substance allowing the replacement or reduction of FCS is desirable.

In this paper a yolk extract (EYF-X) is described originating from chicken eggs which facilitates in vitro culture of a variety of cell types. When the extract was added to a culture medium used for in vitro fertilisation (cat) the amount of successful fertilisations was significantly increased. In a further in vitro model (permanent neuronal cell line N2A) the yolk extract significantly stimulated cell proliferation as well as the growth of cell processes. Surprisingly, a set of antibodies with specificity against different parts of the prepro-cholecystokinin (preproCCK) was able to react with the extract. It could be demonstrated that the intensity of the reaction depends on the egg's age (time after the laying date). Analysis by gel chromatography recorded a main protein fraction with an apparent molecular weight between 20 and 30 kDa. This fraction was labelled by Western-blot using an Ab with specificity against CCK-octapeptid (CCK-8). These findings suggest that the yolk factor may be a CCK/gastrin like molecule.

Since CCK/gastrin-like molecules have been detected also in spermatozoa of mammals the influence on in vitro fertilization could be explained by the yolk factor replacing the endogenous CCK/gastrin-like molecule destroyed in sperm freezing. The results of this study suggest it may be possible to replace FCS by EYF-X but the application of the yolk factor to a broad spectrum of cell types remains to be investigated.

ACKNOWLEDGEMENT

This work was supported by the Zentralstelle zur Erfassung und Bewertung von Ersatz- und Ergänzungsmethoden zum Tierversuch (ZEBET, Grant No.: 13280-150) and partly by the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (Grant No.: 0311470A).