Antibodies used.

The endomembrane system is essential for healthy cell function, with the various compartments carrying out a large number of specific biochemical reactions. To date, almost all of our understanding of the endomembrane system has come from the study of cultured cells growing as monolayers. However, monolayer-grown cells only poorly represent the environment encountered by cells in the human body. As a first step to address this disparity, we have developed a platform that allows us to investigate and quantify changes to the endomembrane system in three-dimensional (3D) cell models, in an automated and highly systematic manner. HeLa Kyoto cells were grown on custom-designed micropatterned 96-well plates to facilitate spheroid assembly in the form of highly uniform arrays. Fully automated high-content confocal imaging and analysis were then carried out, allowing us to measure various spheroid-, cellular- and subcellular-level parameters relating to size and morphology. Using two drugs known to perturb endomembrane function, we demonstrate that cell-based assays can be carried out in these spheroids, and that changes to the Golgi apparatus and endosomes can be quantified from individual cells within the spheroids. We also show that image texture measurements are useful tools to discriminate cellular phenotypes. The automated platform that we show here has the potential to be scaled up, thereby allowing large-scale robust screening to be carried out in 3D cell models.