In vitro co-culture of <i>Fasciola hepatica</i> newly excysted juveniles (NEJs) with 3D HepG2 spheroids permits novel investigation of host–parasite interactions

<i>Fasciola hepatica</i>, or liver fluke, causes fasciolosis in humans and livestock. Following ingestion of vegetation contaminated with encysted parasites, metacercariae, newly excysted juveniles (NEJ) excyst in the small intestine and cross the intestinal wall. After penetrating the liver, the parasite begins an intra-parenchymal migratory and feeding phase that not only drives their rapid growth and development but also causes extensive haemorrhaging and immune pathology. Studies on infection are hindered by the difficulty in accessing these microscopic juvenile parasites <i>in vivo</i>. Thus, a simple and scalable <i>in vitro</i> culture system for parasite development is needed. Here, we find that two-dimensional (2D) culture systems using cell monolayers support NEJ growth to a limited extent. By contrast, co-culture of <i>F. hepatica</i> NEJ with HepG2-derived 3D spheroids, or “mini-livers,” that more closely mimic the physiology and microenvironment of <i>in vivo</i> liver tissue, promoted NEJ survival, growth, and development. NEJ grazed on the peripheral cells of the spheroids, and they released temporally regulated digestive cysteine proteases, FhCL3, and FhCL1/2, similar to <i>in vivo</i> parasites. The 3D co-culture induced development of the NEJ gut and body musculature, and stimulated the tegument to elaborate spines and a variety of surface sensory/tango/chemoreceptor papillae (termed S1, S2, and S3); these were especially pronounced around the oral and ventral suckers that sense host chemical cues and secure the parasite in tissue. HepG2 3D spheroid/parasite co-culture methodologies should accelerate investigations into the understanding of <i>F. hepatica</i> NEJ developmental biology and studies on host–parasite interactions, and streamline the search for new anti-parasite interventions.