Metacyclogenesis and T. cruzi differentiation in vitro

Differentiation of epimastigotes into metacyclic trypomastigotes was accomplished in vitro using chemically defined culture medium⁴⁸. Briefly, epimastigotes in late exponential growth phase (~ 5 × 10⁷ cells ml^-1) in LIT medium were harvested by centrifugation, resuspended in triatomine artificial urine (TAU) medium (190 mM NaCl; 17 mM KCl: 2 mM MgCl:; 2 mM CaCI2; 8 mM phosphate buffer, pH 6.0; 0.035% w/v sodium bicarbonate) at a density of 5 × 10⁸ cells ml^-1 and incubated for 2 h at 28 °C to obtain stressed epimastigotes which were transferred to culture flasks containing TAU3AAG medium (TAU medium supplemented with 50 mM sodium L-glutamate, 2 mM sodium L-aspartate, and 10 mM glucose) at a final density of 5 × 10⁶ cells ml⁻¹ and incubated at 28 °C for 72 h. After this time, the metacyclic trypomastigotes were purified by DEAE cellulose chromatography (D-3764, Sigma), as previously described⁴⁹. Cell differentiation efficiency was determined by counting the different cell types from the supernatant of the 72 h-in vitro metacyclogenesis assay using a hemocytometer. These assays were performed including biological and technical replicates and the data subjected to statistical analysis by unpaired t-test using GraphPad Prism v8 software. Samples from the intermediate forms (stressed epimastigotes and 24 h adhered differentiating forms) and the 72 h differentiated metacyclic trypomastigotes were collected during in vitro metacyclogenesis for subsequent analysis.

Amastigotes were obtained by in vitro amastigogenesis as described elsewhere⁵⁰. Briefly, cell-derived trypomastigotes were cultivated in acidic high glucose DMEM (pH 5.0) at 37 °C for 24 h and the amastigotes collected by centrifugation. Epimastigogenesis to obtain epimastigotes from cell-derived trypomastigotes was performed following a procedure previously described elsewhere⁵¹ with minor modifications. Briefly, cell-derived trypomastigotes were cultivated in LIT (without FBS) at 28 °C for 3 days, then recently differentiated epimastigotes were maintained in culture in LIT supplemented with 10% FBS at 28 °C.