The endomembrane system of mammalian cells provides massive capacity for the segregation of biochemical reactions into discrete locations. The individual organelles of the endomembrane system also require the ability to precisely transport material between these compartments in order to maintain cell homeostasis; this process is termed membrane traffic. For several decades, researchers have been systematically identifying and dissecting the molecular machinery that governs membrane trafficking pathways, with the overwhelming majority of these studies being carried out in cultured cells growing as monolayers. In recent years, a number of methodological innovations have provided the opportunity for cultured cells to be grown as 3-dimensional (3D) assemblies, for example as spheroids and organoids. These structures have the potential to better replicate the cellular environment found in tissues, and present an exciting new opportunity for the study of cell function. In this mini-review we summarise the main methods used to generate 3D cell models, and highlight emerging studies that have started to use these models to study basic cellular processes. We also describe a number of pieces of work that potentially provide the basis for adaptation for deeper study of how membrane traffic is coordinated in multicellular assemblies. Finally, we comment on some of the technological challenges that still need to be overcome if 3D cell biology is to become a mainstream tool towards deepening our understanding of the endomembrane system in mammalian cells.

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