Two-phase in vitro culture of explanted chick embryos

The present study describes a method of culturing chick embryos together with their surrounding area vasculosa on two different culture media in succession. Embryos in the 2nd day of incubation (stages 13, 14, 15 according to Hamburger and Hamilton, 1951) were explanted from the yolk with the aid of a ring of filter paper and transferred dorsal side up to a silicone culture dish containing the first culture medium (89.5% L-15, 10% fetal calf serum, 0.5% Antibiotics). The paper ring was clamped onto the wall of the culture dish by a steel ring so that the embryo was fixed for the culture period. After 4 +/- 1, 8 +/- 1, 12 +/- 1 hrs, the embryos were taken from the culture dishes and transferred to others containing a yolk-albumen mixture as culture medium; 81.2% of embryos survived the first phase of culture. On the second medium 50.3% of explanted embryos were still alive at stage 20 (HH), and 7.9% of them reached the 5th day of development (St 25 HH). The average length of survival in vitro was found to be influenced by both the length of the first culture phase and the stage at which embryos were explanted. This culture method may be useful for teratological tests, since in the first phase of culture, concentrations of test substances and the time of exposure can be exactly adjusted, and in the second phase, the embryo is allowed to develop quite normally, under conditions similar to those in ovo.