Long-term culture and cloning system for Trypanosoma brucei gambiense bloodstream forms in semi-defined medium in...

A new semi-defined medium extremely useful for the long-term cultivation and cloning of Trypanosoma b. gambiense (Wellcome strain) bloodstream forms is described. Bloodstream forms could be continuously grown in 25 mM HEPES-buffered D-MEM supplemented with 10 microM bathocuproine sulfonate (BCS), 100 microM cysteine, and 20% heat-inactivated fetal calf serum at 37 degrees C in vitro. Under these culture conditions, T. b. gambiense bloodstream forms increased in number up to 2-3 x 10(6) trypanosomes/ml by day 3 after initiation of the culture. The trypanosomes maintained in this culture system for 200 days retained their infectivity for mice. Morphologically, they were long and slender, and a surface coat was evident on the cell surface and flagellar membrane. In vitro cloning with single bloodstream forms of T. b. gambiense could be achieved with high efficiency.