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Cellular culture

In cellular media, the entire biological cycle of T. cruzi in the vertebrate host is reproduced. The first culture experiments were with tissue fragments absorbed with coagulated plasma or pendant drop.114,115 Further monolayer culture of cell lineages regularly maintained in the laboratory was used. Cells of various tissues and organs such as heart, kidney, lung, skin, and skeletal muscle can be used. Metacyclic try- pomastigotes originating from culture and vector, as well as bloodstream forms of the parasite are able to infect cells in culture although it has been demonstrated that vector trypomastigotes are more infective than the metacyclic forms of cul- ture.116,117 Inside these cells, the parasite transforms into amastigotes, which after some time or several cell generations differentiate into new trypomastigotes that

will be released in the extracellular medium and be able to invade new cells. With the cultivation of the cells in liquid medium containing mammal sera, several lineages of cells were adapted for T. cruzi culture. Today several different cell lineages are used for T. cruzi cultivation.

Cryopreservation

T. cruzi, as well as other microorganisms, can be preserved for long periods of time in liquid nitrogen (— 196°C) without changing its original characteristics. Filardi and Brener118 were the first to systematically study T. cruzi cryopreservation and verifying its effect on several biological characteristics of the parasite (infectivity to vertebrate and invertebrate hosts; morphology, parasitemia, and mortality in mice). These authors verified that initially blood trypomastigotes and epimastigotes obtained from artificial cultures need to be mixed with an equal volume of glycerin 10% and maintained at —73°C for 16—20 h before cryopreservation in nitrogen. Later these same authors119 verified that exemplars of triatomine vectors infected with T. cruzi can also be cryopreserved with preservation of parasite characteristics including its infectivity to vectors. Dimethyl-sulfoxide (DMSO) 5—10% is also used as a cryoprotective agent for trypanosomes. However, it was demonstrated later120 that cryopreservation and thawing of T. cruzi may lead to severe damage of the mitochondrial apparatus and thus to severe disorders of metabolic function, exhaustion of the metabolic pool, and finally to death of the damaged trypanosomes, despite the use of DMSO as a cryoprotective agent. Yager121 demonstrated that tissue homogenates containing T. cruzi amastigotes or Leishmania spp. were also rapidly frozen with 10% glycerol as cryoprotectant and the viability and pathogenicity of the parasites maintained for several years.

Cryopreservation made it possible to create banks of T. cruzi strains with parasites from around the world, which was very important to several studies on various aspects of this parasite and provided a better idea of its polymorphism. Moreover, cryopreser- vation avoids biological changes in its original characteristics induced by selection throughout long-term maintenance in the laboratory in different conditions, such as successive passages in mice, the risk of accidental infections in the laboratory, and the comparative study of a large number of parasite populations of distinct origins.

Successive and alternative passages in animals and vectors

These alternatives are very frequently used in the laboratory to maintain the parasite for experimental studies. Murine models were the most widely used models for this purpose because they are very susceptible to infection and easy to reproduce and maintain in the laboratory. Parasite populations are maintained in this animal model by successive reproduction of the acute phase of the infection. Moreover, T. cruzi can also be maintained by successive passages in triatomines and alternative passages through tria- tomine vectors and laboratory animals. This type of maintenance has been considered to be very important to prevent the biological behavior of the parasite observed in the laboratory after long-term successive passages in animal models or culture.

 
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