Optimization of Entamoeba histolytica culturing in vitro

Entamoeba histolytica is among the most deadly parasites accounting for the second highest mortality rate among parasitic diseases. Nevertheless, contrary to trypanosomatids, this protozoan in hardly studied by parasitology groups. This astonishing discrepancy is largely due to the remarkable intricate conditions required for parasite proliferation in vitro, particularly whenever large cell numbers are required. The present study was undertaken in order to optimize E. histolytica culturing harvest, using mineral oil layers preventing culture medium-air contact to maintain anaerobic conditions in culture plate wells. 2×10⁴ trophozoites were plated on each well in 2.0mL YI-S-33 medium, supplemented with bovine serum and 700μL mineral oil. Parasites were daily quantified by light microscopy counting for up to 96h and trophozoite motility was also assessed. We notice that E. histolytica cultures in 24-well plates reached several-fold higher cell densities, particularly whenever the mineral oil layer was placed on top of the medium surface, blocking the air interface. At least 99% of the parasites were vigorously motile for 72h in oil-containing wells, whereas only less than 5% displayed significant motility in oil-devoid wells. In order to determine whether such different growth responses were due at least in part to the oxidative stress, we used the reactive oxidant species fluorescent probe dihydroethidium (DHE). The remarkably higher DHE parasite labeling in oil-devoid cultures indicate that oxidative stress reduction can play a significant role in elevated growth rates observed in oil supplemented cultures. Propidium iodide and Trypan blue dye-exclusion assays indicate that parasite necrosis resulted from the stressing conditions. The present study indicates that E. histolytica culturing in oil-sealed wells may comprise a valuable tool for bioactivity of antiparasitic compounds.