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Three-dimensional (3D) tissue models such as epithelial spheroids or organoids have become popular for pre-clinical drug studies. In contrast to 2D monolayer culture, the characterization of 3D tissue models from non-invasive brightfield images is a significant challenge. To address this issue, here we report a deep-learning uncovered measurement of epithelial networks (Deep-LUMEN) assay. Deep-LUMEN is an object detection algorithm that has been fine-tuned to automatically uncover subtle differences in epithelial spheroid morphology from