Optimizing In Vitro Metacyclogenesis: Strain-Specific Variability in Trypanosoma cruzi Responses to Nutritional and pH Stress

Metacyclogenesis is a critical process in the Trypanosoma cruzi life cycle. This transition naturally occurs in an insect vector’s gut. Although Triatomine Artificial Urine (TAU) medium is the widely adopted approach to metacyclogenesis in vitro, its efficiency decreases with prolonged epimastigote culture, limiting the availability of metacyclic trypomastigotes for study. We aimed to establish a practical and efficient method for generating high concentrations and purities of metacyclic trypomastigotes in vitro. Epimastigotes of the Dm28c strain were exposed to pH shifts in nutrient-rich (MT-LIT) and nutrient-poor (M16) media, under static or agitated conditions. Both media promoted higher metacyclogenesis rates than TAU, with epimastigotes’ adherence to the substrate being a crucial factor. Metacyclogenesis efficiency varies depending on the strains and culture conditions. Notably, both LIT and M16 at pH 6 produced metacyclic trypomastigotes with infective capacity on Vero cells. Under these conditions, a variety of intermediate forms were observed compared to those induced by TAU metacyclogenesis. Our findings further emphasize the strain-dependent nature of optimal in vitro metacyclogenesis conditions and offer new opportunities for studying the intermediate forms involved in this essential process.