Standard culture medium allows clonal dilution of Trypanosoma brucei procyclic cells after auto-conditioning

Trypanosoma brucei can be cultured in vitro in the mammalian bloodstream form or in the procyclic (PC) form found in the insect vector. Bloodstream trypanosomes can be cloned by limiting dilution, but PCs can only be diluted in conditioned medium, i.e., medium in which PC cells have previously been grown. It is shown here that this limitation does not apply to the most commonly used PC cell strain, Lister 427, if free radicals are removed from the medium. The reported benefit of conditioning media may arise in part from a process of hemin-catalysed depletion of peroxide (“auto-conditioning”) which occurs during extended incubation at growth temperature. Scavenging free radicals by addition of pyruvate also improves PC cell viability. However, other PC cell strains such as Treu 927 require cell-conditioned media unless grown in a 5% CO2 atmosphere. Several other culture parameters that affect growth rates and dilution capability were identified.