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Maintaining T. cruzi in the laboratory

T. cruzi can be cultivated and maintained in the laboratory in several conditions: acellular/axenic cultures, cellular cultures, successive passages in different laboratory animal models (especially mice), alternative passages through vertebrate (mice or other experimental models) and invertebrate hosts (triatomine vectors), and cryopreservation.

Acellular culture

Acellular culture reproduces the biological cycle of T. cruzi developed in triatomine vectors. The first cultures of T. cruzi were in acellular biphasic or monophasic

medium. The monophasic medium offers advantages because the parasite can be obtained with less contaminant and parasite growth can be assessed by cell counting using an electronic device. The most important and most widely used were liver infusion-tryptose (LIT) and Warren media. Camargo102 was the first to describe the cellular transformations of T. cruzi and growth in LIT medium, the most useful monophasic medium used in the laboratory. Several important studies on metacyclogenesis in LIT media have been described. It was also demonstrated that in the medium called M16, nutrient-poor with low pH, a higher percentage of metacyclogenesis is obtained, although the type of T. cruzi strain considered is also important.

The semidefined and defined media for trypanosomes were described byYoshida103 and Roitman et al.104 Chemically defined TAUP104 or TAU3GAA106 media using components similar to vector urine were used for better differentiation in vitro. The existence of culture media free of macromolecules, especially the chemically defined medium, provides the large quantity of parasite cells necessary for antigen preparations, biochemical studies on nutritional requirements, metabolic pathways, and molecular characterization. Using the semidefined medium 4 as overlay and a monophasic medium using blood of different animal species, high rates of T. cruzi and Leishmania donovani growth were obtained by Perlewitz and Koch,107 which improves the chance of obtaining isolations of these parasites.

Bonaldo et al.108 verified that nutritionally poor medium promotes the metacyclogenesis of T. cruzi. The results of Duschak et al.109 indicate the presence of a novel cysteine proteinase secreted by metacyclic trypomastigotes and reinforces the important role played by these enzymes in T. cruzi metacyclogenesis. Interestingly, biological changes in the parasite’s infectivity and metacyclogenesis have been observed after successive passages in acellular culture, which can be restored after maintenance by successive passages in triatomines.110 De Lima et al.111 verified that cultivation of T. cruzi epimastigotes in low-glucose axenic media shifts its competence to differentiate at metacyclic trypomastigotes. Differential gene expression for different periods (6 and 24 h) of T. cruzi metacyclogenesis was observed by Krieger et al.112 and a proteomic analysis113 identified relevant proteins involved in the metacyclogenesis process. Their identification and molecular characterization is highly important in understanding the steps of parasite differentiation into the infective form.

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