Cysteine eliminates the feeder cell requirement for cultivation of Trypanosoma brucei bloodstream forms in vitro

In all previous studies, bloodstream forms of Trypanosoma brucei could be grown in vitro only when supported by a feeder layer of mammalian fibroblasts. We have axenically cultivated bloodstream T. brucei by adding L-cysteine at regular intervals and appropriate concentrations. The optimum cysteine concentration depends on cell density and is close to physiological serum levels. At concentrations >24mg/liter (2 x 10(-₄) M), cysteine was acutely toxic to trypanosome concentrations of 3 x 10₍₇₎/ml. Toxicity was prevented by addition of pyruvate or catalase, which neutralize H₂O₂ produced by cysteine autoxidation. In uptake studies using [₃₅S]cysteine and [₃₅S]cystine, T. brucei efficiently incorporated only cysteine. The K(m) for cysteine uptake was 4 x 10_(-4) M. Cystine supported axenic growth if low concentrations of 2-mercaptoethanol were added at regular intervals.