Abstract
Three-dimensional (3D) cell culture models bridge the gap between two-dimensional (2D) monolayer cultures and animal models. Physiologically relevant, 3D culture models have significantly advanced basic cell science and provide unique insights into host-pathogen interactions intrinsically linked to cell morphology. Toxoplasma gondii is an obligate intravacuolar parasite that chronically infects a large portion of the global human population. Our current understanding of Toxoplasma infection is largely based on 2D cell cultures, in which mammalian cells are grown on flat surfaces. However, 2D cell cultures may not recapitulate key conditions of in vivo infections as they introduce artificial pressures and tensions, which may subsequently alter infectious processes that are dependent on spatiality, e.g., invasion, replication and egress. In this study, we adapted a collagen-based 3D cell culture system to reproduce the 3D environment of T. gondii natural infections for investigation of the replication and egress of the parasite from the parasitophorous vacuole. Suspended in the 3D matrix, Toxoplasma-infected VERO cells have round morphology, as opposed to infected VERO cells in 2D monolayers. The doubling time of Toxoplasma in VERO cells within the matrix is comparable to that of parasites cultivated in VERO cell monolayers. In the absence of the pressure of flattened host cells grown in 2D cultures, the parasitophorous vacuole of T. gondii has a globular shape, with intravacuolar parasites distributed radially, forming 3D spherical ‘rosette’ structures. Parasites egress radially away from the ruptured host cell in 3D matrices, in contrast to Toxoplasma cultivated in 2D monolayer cultures, where the parasites escape perpendicularly from the flat surface below the host cells. These observations demonstrate the utility of collagen matrices for studying parasite modes of infection as these 3D assays more closely mimic in vivo conditions.
Highlights
We used collagen fibers to reproduce an extracellular-like matrices (ECM)-like environment for the 3D cultivation system of cells infected with T. gondii to compare parasite processes in 3D and 2D cell culture models
We selected human foreskin fibroblasts (HFF) as host cells for Toxoplasma in our 3D cultivation system since HFF are routinely used in many laboratories for 2D culture as they grow as large and flat cells that are contact-inhibited
In our 3D culture system, infected cells had a rounder morphology, parasitophorous vacuole (PV) were more globular in shape and the parasites organized themselves into spherical ‘rosettes’ within the PV
Summary
Investigations on T. gondii parasitism have been divided between in vitro, such as 2D cell cultures, and animal infection models. The structural constraints of growing cells on flat surfaces in culture dishes yield cells with artificial shapes and different architecture than in animal tissues; it does not recapitulate natural infections. Animal models are exploited as tools to investigate cyst burden and distribution in tissues, immune responses, and pathogenicity of chronic infections. The pioneering of a bioluminescence system in T. gondii (luciferin/luciferase), in addition to other genetic modifications of the parasite, has allowed the monitoring of the spread of an active infection in living animals [6]. Animal models are time- and money-intensive, they give very little experimental precision on individual infections or do not permit the examination of parasite-host cell interactions at the subcellular level
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