Short communication: cultivation of bloodstream forms of Trypanosoma brucei and T. evansi in a serum-free medium

Bloodstream forms of Trypanosoma brucei and T. evansi have been cultivated in an axenic culture system by using Iscove's modified DMEM-based HMI-9 medium supplemental with bathocuproinedisulphonic acid, L-cysteine, hypoxanthine, 2-mercaptoethanol, pyruvate, thymidine and 10% fetal bovine or adult horse serum. We developed a serum-free medium (HMI-244) in which serum in HMI-9 was replaced by fatty acid-free bovine serum albumin, bovine alpha 2-macroglobulin, bovine beta-lipoprotein, d-biotin, retinol, beta-alanine, L-anserine nitrate salt, L-ornithine hydrochloride, O-phosphorylethanolamine, sarcosine, taurine, adenosine-5'-triphosphate, 2'-deoxycytidine-5'-monophosphate, 2'-deoxyguanosine-5'-monophosphate, and 5-methyltetrahydrofolic acid. Maximum cell densities and population doubling times were 2.6 x 10(6) cells/ml; 10.6 hours and 2.2 x 10(6) cells/ml; 10.9 hours for T. brucei and T. evansi, respectively. Bloodstream forms continued to proliferate in the serum-free cultures for more than 90 days and the trypomastigotes retained their morphological characteristics and infectivity to mice. If validated, this serum-free medium may help reduce future interlaboratory variability in biochemical, immunological, molecular biological and drug sensitivity studies on these parasites.