Lessons on multicellular two- (2D) and three-dimensional (3D) culture in parasitology: Insights, challenges and...

Advances in multicellular two-dimensional (2D) and three-dimensional (3D) cell culture systems are providing parasitologists with new tools to investigate host-parasite interactions in vitro. These models offer tissue-specific and, increasingly, host-specific alternatives to traditional 2D monoculture and animal systems, with applications across protozoan and helminth biology. Spheroids, organoids, and emerging assembloid platforms capture key aspects of tissue architecture and function, enabling co-culture of parasites and their products, including dynamic analysis of interactions at defined host interfaces. In recent years, these systems have been adapted to model infection processes, parasite development, immune modulation and tissue remodelling across a range of parasite taxa and tissue types, particularly of the gastrointestinal tract. This review outlines applications of multicellular 2D and 3D cell culture systems in parasitology, drawing on examples from both human and veterinary research. We highlight lessons learned from published works to date that have accelerated the uptake and refinement of these approaches. We also examine technical challenges, including issues of standardisation, scalability, model accessibility, and species representation, particularly for livestock hosts. Looking ahead, the integration of immune, stromal, and microbial components into these models, as well as advances in imaging and omics technologies, and CRISPR-Cas9-mediated engineering of host organoids, promise increasingly sophisticated platforms for studying parasite biology, host tissue responses and pathogenesis. With continued investment and cross-disciplinary collaboration, multicellular culture systems are poised to play a central role in reducing animal use, improving model predictiveness, and supporting the development of next-generation antiparasitic therapies and interventions, including drugs and vaccines.