Designing a 3D cell culture system for the in vitro investigation of Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite present in terrestrial and marine biomes around the world and highly prevalent in human populations, causing chronic infections. In immuno-compromised individuals, this pathogen can cause fatal toxoplasmosis and in pregnant women abortion of the fetus. Although Toxoplasma poses serious public health risks, there are few well-tolerated and efficient drugs to treat the parasite, and no drugs completely clear the parasite from the infected host. Research performed on Toxoplasma in 2D monolayers disregards the problem that 2D cell culture cannot recapitulate key conditions of in vivo infections. 2D cell culture introduces artificial pressures and tensions as well as altering normal infectious processes, such as replication and egress. In order to solve these problems while still maintaining an in vitro environment, we adapted a collagen-based 3D cell culture system to recapitulate the 3D environment of in vivo infections. We used collagen to simulate the extracellular matrix, creating 3D collagen matrices that replicated the density of tissue (2 mg/mL). Our data show that in our 3D system of VERO cell cultivation, the cells exhibit similar viability to their 2D counterparts. In 3D conditions, Toxoplasma replicates efficiently, at a similar rate as parasites in 2D monolayers. We observed globular parasitophorous vesicles and spherical ‘rosette’ formation of parasites within the vacuole. Furthermore, Toxoplasma infection in 3D changes the phenotype of the host cell and parasite egress from cells is characterized by the escape of free parasites in all directions away from the cell. We also recapitulated host mitochondria association with parasite PVs in 3D suspended cells. In conclusion, it is certain that parasite processes are best recapitulated in a 3D environment. This technique is particularly critical for processes that are dependent on spatiality, such as Toxoplasma invasion, replication, egress