In the lab where I work, it's used the experimental protocol of Canavaci et al (2010) in order to elicit metacylogenesis in epimastigotes from CL Brener strain cultivated in LIT medium. Then, these morphology types are incubated for 2 hours in TAU medium to them attach in the flask wall. After that, the medium is discarded and replaced with TAU3AAG for 6 days. Parasites are incubated again from recovering of supernatant into fetal bovine serum in order to lyse the epimastigotes forms and, in this sense, obtain metacyclic trypomastigotes. However, by this method I've noticed that only a maximum of 64%, and usually about 31% of metacyclic trypomastigotes are obtained.
In this way, I would like to know: Is there another way of obtaining more metacyclic trypomastigotes in order to improve this protocol?
In vitro transformation of metacyclic trypomastigotes into amastigotes - At 37°C, MEM 0.4% BSA medium has been reported to induce 100% of bloodstream try-pomastigotes into amastigotes (Tomlinson et al. 1995). However, under these conditions metacyclic try-pomastigotes failed to produce amastigotes, and after one day of incubation all the parasites lysed. After testing different media and incubation conditions, we could obtain a high yield of differentiation forms and EMA at 37°C in a 5% CO2 atmosphere. This was achieved in two steps: Step 1 consisted of the pre-incubation of metacyclic trypomastigotes at 37°C in MEMTAU for three days. The metacyclic trypomastigotes underwent very few morphological changes, and a high percentage of well-preserved metacyclic trypomastigotes (up 80%) was obtained; Step 2: the subpopulation of untransformed metacyclic trypomastigotes was purified through DEAE-52 columns (I = 0.145), and when these purified metacyclic try-pomastigotes were incubated again in fresh MEMTAU medium at 37°C, a high percentage (up 80%) of typical, well-preserved amastigotes were progressively produced. The in vitro-derived amastigotes did not display any detectable signs of degeneration and were indistinguishable from normal amastigotes.
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