In vitro expansion of T. gondii tachyzoites using a magnetic aggregation 3D cell culture

Abstract

Toxoplasma gondii is a protozoan parasite capable of infecting a wide range of living beings, including felines, which are the definitive hosts of toxoplasmosis, as well as livestock, birds, and fish. In humans, the parasite can also be present in a latent or cystic form, which may become chronic and lodge in the brain, retina, or muscles. Infection occurs upon consuming water or food contaminated with oocysts. Tachyzoites of the RH strain have fast replication and are relatively difficult to maintain exclusively in vitro, often requiring stages of in vivo cultivation in experimental animals. Three-dimensional nanostructured cell cultures can be useful to develop new forms of in vitro production with potential gains in practicality and yield. This work aimed to demonstrate the feasibility of using a three-dimensional culture of murine fibroblasts aggregated with nanoparticles as a substrate for T. gondii tachyzoites, with the intention of facilitating the maintenance and in vitro replication of the parasite. Magnetic aggregation was used to produce cell spheroids, which were infected with tachyzoites of the RH strain and maintained in culture. After infection, spheroids were evaluated by transmission electron microscopy and fluorescence microscopy with 3D rendering of image stacks. The presence of the parasite was confirmed by PCR, and the number of free parasites in culture was evaluated by flow cytometry. The three-dimensional culture model used showed sustainable production of tachyzoites within 24 hours after inoculum, demonstrating potential as a surrogate for the use of animals for parasite maintenance.