ATLA::Alternatives to Laboratory Animals

Volume 28, Number 6

Replacement of fetal calf serum in cell cultures by an egg yolk factor with cholecystokinin/gastrin-like immunoreactivity.

ATLA 28, 815-831, September/October 2000

Mirko Sasse,¹ Thomas Lengwinat,² Peter Henklein,³ Andreas Hlinak⁴ and Rüdiger Schade

¹Institute of Pharmacology and Toxicology, Medical Faculty (Charité), Humboldt-University, Dorotheenstrasse 94, 10117 Berlin, Germany; ²Institute of Zoo and Wildlife Biology, PF 601103, 10252 Berlin, Germany; ³1nstitute of Biochemistry, Medical Faculty (Charité), Humboldt-University, Hessische Str. 3-4, 10115 Berlin, Germany; ⁴State Veterinary and Food Investigation Centre, Ringstrasse 1030, 15234 Frankfurt/Oder Germany

SUMMARY

The in vitro culture of various cell types is an important scientific tool and is becoming increasingly acceptable as a viable alternative to animal experiments. Fetal calf serum (FCS) is a supplement used in many cell culture media, and provides cells with growth factors and cytokines necessary for successful culture. In view of the animal welfare issues surrounding the production of FCS, an alternative agent allowing the replacement or reduction in the use of FCS is desirable. A yolk extract factor, egg yolk factor X (EYF-X), obtained from chicken eggs is described, which facilitates the in vitro culture of a variety of cell types. When the extract was added to a culture medium used for in vitro fertilisation, the number 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. A set of specific antibodies against different parts of the prepro-cholecystokinin reacted with the extract. The intensity of the reaction depended on the age of the egg (time after the laying date). Analysis by gel chromatography recorded a main protein fraction with an apparent molecular mass of 20-3O kDa. This fraction was labelled by Western blot with an antibody with specificity against CCK-octapeptide. These findings suggest that the yolk factor may be a CCK/gastrin-like molecule. Since CCK/gastrin-like molecules have also been detected in the spermatozoa of mammals, the influence on in vitro fertilisation could be explained by the yolk factor replacing the endogenous CCK/gastrin-like molecule destroyed in sperm freezing. The results of this study suggest that it might be possible to replace FCS with EYF-X. The application of the yolk factor to a broad spectrum of cell types remains to be investigated.

Keywords: fetal calf serum, chicken egg yolk factor, cholecystokinin/gastrin-like immunoreactivity, in vitro fertilisation, permanent neuronal cell culture, growth stimulation