Digitalized organoids: integrated pipeline for high-speed 3D analysis of organoid structures using multilevel segmentation and cellular topology.

Organoids replicate tissue architecture and function and offer a unique opportunity to explore the impact of external perturbations in vitro. However, conducting large-scale screening procedures to investigate the effects of various stresses on cellular morphology and topology in these systems poses important challenges, including limitations in high-resolution three-dimensional (3D) imaging and accessible 3D analysis platforms. In this study, we introduce an AI-based multilevel segmentation and cellular topology pipeline for screening morphology and topology modifications in 3D cell culture at both the nuclear and cytoplasmic levels, as well as at the whole-organoid scale. We demonstrate the versatility of our approach through proof-of-concept experiments, encompassing well-characterized conditions and poorly explored mechanical stressors such as microgravity. By offering a user-friendly interface named 3DCellScope and a comprehensive set of tools for discovery-like assays in screening 3D organoid models, our pipeline demonstrates wide-ranging potential for applications in biomedical research.