Effective methodology for maintaining Toxoplasma gondii in vitro using paramagnetic iron nanoparticles to support three-dimensional cell culture

Abstract

Toxoplasma gondii is a protozoan parasite that infects approximately one billion people worldwide. Upon infection, the host may die due to latent infection or presence with chronic cysts in brain, retina or muscle tissue. Humans can become infected consuming water or foods contaminated with oocysts or eating undercooked meat. Its virulent form is difficult to replicate in vitro, requiring additional steps using experimental animals. The use of nanotechnology can contribute to this in vitro production, through the three-dimensional cultivation of mouse fibroblast cells (NIH/3T3 ATCC® CRL-1658™) and nanoparticles synthesized with radiation. The objective of this work was to demonstrate the three-dimensional culture of fibroblast cells aggregated to nanoparticles for inoculation the T. gondii. This methodology was created to facilitate parasite management and replication. For the production of nanoparticles, the work used concentrations of iron sulfate II heptahydrate (Fe2SO4.7H2O, CAS 7782-63-0) and glycine (NH2CH2COOH, CAS 56-40-6) diluted in ultrapure water free of O2 at pH 12. This solution was irradiated by electron beam of the IPEN / CNEN-SP Radiation Technology Center in doses of at least 15 and at most 30kGy. Paramagnetic iron oxide nanoparticles (PION’s) were then adsorbed on cell membranes, and cells were kept together by a magnetic field. Structured spheroids (4 day of culture) were infected with 106 parasites (RH strain) and the infection was evaluated by transmission electron microscopy. Tachyzoites were found inside 3T3 cells, assuring that the spheroid can be a suitable culture substrate to T. gondii in vitro propagation. A three-dimensional methodology for in vitro cultivation of the parasite is perhaps the key for applications in the study of toxoplasmosis, as it has a fast, cheap, efficient production (yield and reduction of contamination).