Toxoplasma gondii is a protozoan parasite capable of infecting a wide range of living beings, including felines that are the definitive hosts of the disease, toxoplasmosis, and livestock, birds and fish. In humans, the parasite can also be present in a latent or cystic form, the latter being able to become chronic, leading to lodging in brain, retina or muscles. Infection occurs upon consuming water or food contaminated with oocysts. The tachyzoites of RH strain have fast replication and relative difficulty of maintanence exclusively in vitro, often requiring stages of in vivo cultivation in experimental animals. Three-dimensional nanoestrutucured cell cultures can be helpful to build new forms of in vitro production with potential gains in practicality and yield. This work aimed to demonstrate the feasibility of use of three-dimensional culture of murine fibroblasts aggregated to nanoparticles as substrate for T. gondii tachyzoites with the intention of facilitating the management and in vitro replication of the parasite. Magnetic aggregation was used to produce cell spheroids, which were infected with tachyzoites of 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, showing itself as a potential surrogate for the use of animals for the maintenance of the parasite.