Patient-Derived 3D Cell Models as New Approach Methodologies: Applications, Advances, and Challenges for...

Patient-derived three-dimensional (3D) cell models are new approach methodologies (NAMs) that bridge the gap between traditional 2D cell cultures and in vivo models, making them attractive for increased-throughput screening (ITS) and secondary assays. In this review, we outline screening applications implementing patient-derived 3D cell models, with an emphasis on patient-derived organoid (PDO) based assays, across oncology, toxicity testing, and rare disease drug discovery. We summarize how tumor and non-tumor organoids are used to discover new drug vulnerabilities, prioritize standard-of-care regimens, and model dynamic pathophysiologic processes in 96 to 1536-well formats. We then highlight advances in plating strategies, assay design, and advanced assay outputs that have enabled semi-automated and fully automated pipelines. Finally, we discuss persistent challenges, including heterogeneity, assay standardization, and limited publicly available protocols, and propose future directions such as integration with immune and stromal compartments, and national standardization efforts to increase reproducibility and translational impact.