A Robust Protocol for the Establishment, Expansion, Bio-Banking, and Molecular Profiling of Triple-Negative Breast Cancer Organoids

Patient-derived organoids (PDOs) offer a powerful in vitro platform to model cancer biology, investigate therapeutic responses, and personalize treatment strategies. Here, we describe a comprehensive and reproducible protocol for establishing 3-dimensional (3D) organoid cultures from surgically resected triple-negative breast cancer (TNBC) samples. Fresh TNBC surgical tissues were processed using a standardized enzymatic and mechanical dissociation protocol followed by 3D culture in Matrigel. Organoids were expanded, cryopreserved, and characterized through histology, immunohistochemistry (IHC), and whole-exome sequencing (WES). Immunophenotypic and genomic concordance was assessed between PDOs and their paired tumors. Organoids were successfully established from resected TNBC tissues, displaying histologic, immunohistochemical, and genomic fidelity supporting high-throughput and personalized research applications.