The successful system in long-term cultivation of Trypanosoma gambiense bloodstream forms.

Rat astroglioma cell line (GA-1) was extremely useful for long-term in vitro cultivation of Trypanosoma gambiense blood-stream forms. Parasites could be continuously grown at 37 degrees C for more than 200 days in the culture system, consisted of HEPES-buffered RPMI 1640 (pH 7.2, 300 milliosmole/kg) supplemented with 20% inactivated fetal calf serum in the presence of GA-1 cells. Parasites cultured for more than 200 days still retained not only their virulence for mice but also their original antigenic type. Morphologically, they resembled host infected bloodstream forms by way of having a subterminal kinetoplast and surface coat. The best growth rate of trypanosomes was obtained with 1 X 10(6) GA-1 cells/25 cm2 culture flask. Under this culture condition trypomastigote form populations increased in number up to 7-8 X 10(6) trypanosomes/ml by day 3 after initiation of the culture. The population doubling time in this culture system within the first 24 hours was almost the same as in mice. Most of the cultured trypanosomes were in suspension, but 15-20% of the parasites adhered to the surface of GA-1 cells. The culture system was also shown to be useful for cloning of T. gambiense which is important for separation of mutants.