View in optical microscope of the various developmental stages of
This chapter aims to present and discuss the main cell culture techniques used for the growth and maintenance of the different evolutionary forms of the protozoan Trypanosoma cruzi, the etiologic agent of the Chagas disease. Chagas disease is a neglected tropical disease endemic in several Latin American countries. Here, we intend to present the main difficulties, advantages, and disadvantages of using Trypanosoma cruzi cell culture in parasitic biology. Finally, we present the main research opportunities in the context of Trypanosoma cruzi parasitic biology using cell culture techniques, such as drug development, characterization of pharmacological targets, molecular markers for diagnosis, structural biology, and many other biomedical applications.
- Trypanosoma cruzi
- Chagas disease
- cell culture
- in vitro assay
Carlos Chagas described the American trypanosomiasis through microscope observation of hemoflagellate protozoan in the sample blood and named
|Localization||Intestine of the vector and axenic culture||Rectal ampoule of vector axenic culture||Blood and intracellular spaces of vertebrate-host and cells culture||Inside the cells of vertebrate-host and cells culture|
|Position of the kinetoplast||Near the nucleus||After the nucleus||After the nucleus||Near the nucleus|
Due to its genetic diversity, the parasite
A century of its discovery, Chagas disease remains a cause of early mortality and morbidity in individuals at productive age in endemic areas, generating significant socioeconomic impact. The importance of parasite biology studies is undeniable. In this context, in vitro studies of
2. How to maintain and cultivate
During life cycle,
Substantial interest in understanding the cell biology of
The axenic cultures of
Epimastigote forms are maintained in axenic conditions at 28°C, in the nutritive medium liver infusion tryptose (LIT), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) as described by Camargo . Under chemically defined conditions and suppression of nutrients, the transformation of epimastigotes into metacyclic trypomastigotes is possible in vitro using a process named metacyclogenesis .
To obtain intracellular amastigote forms, usually mammal cells, Vero lineage, murine peritoneal macrophages, fibroblast (mouse L929), and primary heart or skeletal muscles cells are commonly incubated with specific medium supplemented with 10% inactivated FBS (v/v) and allowed to adhere usually for 24 h at 37 ± 2°C in 5% CO2. After this period, adherent cells can infect with trypomastigote metacyclic using a 10:1 ratio of parasites per cell and incubated at 37°C in 5% CO2, without FBS. Amastigote can be observed in microscope at 4–24 h after infection. Intracellular amastigote can release within 72 h trypomastigote metacyclic from culture derived. Infected Vero cells can be in continuous cultivation during 6–8 weeks .
Infected cells with
3. In vitro differentiation of
Trypanosoma cruziepimastigote to trypomastigote forms
Metacyclic trypomastigotes (non-replicative) originating from the insect vector or trypomastigotes from infected cells, such as culture derived and bloodstream/tissue, are infective forms of the parasite. Metacyclogenesis is observed in the stationary phase of culture, and metacyclic trypomastigotes are largely absent during logarithmic growth. However, in vitro differentiation of
By contrast, chemical modification is commonly used for differentiation of epimastigote to trypomastigote forms. Epimastigote forms can be collected from the LIT culture, during the stationary phase of the growth curve, and then resuspended in artificial triatomine urine (TAU) supplemented with proline (TAUP medium) or TAU supplemented with L-proline, L-sodium glutamate, L-sodium aspartate, and D-glucose (TAU3AAG medium) . In the first medium, parasite culture can differentiate after 10 days and 72–96 h using TAU3AAG medium. Recent study demonstrated that reductive environment using urate, a salt or ester of uric acid, could promote epimastigote differentiation with significant increment of trypomastigotes . In addition, Grace medium supplemented with
Metacyclic trypomastigotes are essential for the understanding of host-pathogen interaction and it is remarkable genetic variability between strains. Although, these biological stages in axenic medium are difficult to purify, culture remains with epimastigote stages without showing a high efficiency in the purification of metacyclic trypomastigotes .
Complement-mediated lysis of epimastigotes followed by separation of the trypomastigotes by gradient centrifugation through a dense albumin column represents a selective method that exploited to purify viable trypomastigotes from cells culture medium. Alternate pathway activation seems capable by itself generating epimastigote lysis and consequent activation of the classical pathway just in the presence of serum .
Other several techniques such as density separation with Percoll [18, 19] and extensive use of chromatography have permitted purifying metacyclic trypomastigotes for the association of specific molecules expressed on this parasite stage membrane separation. Chromatography based on the differential plasma membrane charge between epimastigotes and trypomastigotes using ion exchange chromatography with resins, such as cellulose-DEAE and sephadex, have been developed for the purification of metacyclic trypomastigotes [20, 21]. These previous chromatography techniques presented a major disadvantage such as the decrease in the infective capacity of the obtained trypomastigote .
Recently, a new protocol in LIT medium cultures was implemented using sepharose-DEAE as a resin during purification process, which could recover metacyclic trypomastigotes for two different DTUs (I and II). DTU TcI was the one recovered a greater amount of these forms, and parasite infectivity in vitro and in vivo was maintained . Metacyclogenesis with different DTUs exhibited significantly different morphologies and metacyclogenesis pattern .
Still it remains crucial to have easy, fast, and reliable tools to obtain purified metacyclic trypomastigote forms of
4. Advantages and limitation of in vitro
Probably, the main advantages of the use of in vitro systems for maintenance and cultivation of
As mentioned above, the use of
One of the possibilities of in vitro system is to obtain large amounts of metacyclic trypomastigotes (derived culture or chemical modification medium) with biological properties similar to those of the insect-derived forms, which facilitates the study of the biology of
The great potential of the in vitro models is the study of the interaction
However, the in vitro assay does not provide full information on the behavior and characteristics about
5. Applications in the cell culture of
Despite decades of research in
In this chapter, we have shown the importance of cell culture techniques as a complementary tool for studies of parasitic biology. Through the cultivation of different evolutionary forms of
CJGM, JWFO, and AMQ are grateful for the financial support provided by Capes/Brazil through postgraduate scholarships. MSS thanks Global Health and Tropical Medicine, Lisbon-Portugal (GHTM-UID/multi/04413/2013), and to Berenice Project supported by FP7 European Union (grant number 305937). We are also grateful to Paulo Fanado for editing this manuscript.
Conflict of interest
The authors declare no conflict of interest.