Optimization of tumor tissue enzymatic dissociation for rapid and viable tumoroid generation

Patient-derived tumoroids, or 3D organoid cultures from dissociated tumor tissue, are a powerful translational tool that bridge molecular profiling with functional drug testing. However, the success of tumoroid derivation is critically dependent on preanalytical variables-tissue handling, transport, and enzymatic dissociation-that influence viable cell recovery and culture performance. Here, we report an optimized workflow and enzyme formulations to maximize cell yield and fitness for downstream tumoroid culture and functional assays.Fresh surgical resections from colorectal, endometrial, breast, lung, and head and neck cancers were processed to assess the impact of dissociation on downstream culture. Tissues were collected in Hibernate™-A-based transport buffer supplemented with GlutaMAX™ and B-27™ supplements, antibiotics, and the ROCK inhibitor Y-27632, to ensure stability during overnight transport on ice. Upon receipt, samples were washed in Advanced DMEM/F-12-based tissue processing buffer and mechanically minced before enzymatic dissociation. A designed experimental framework evaluated enzyme type, concentration, and interactions across donors, measuring outcomes of viable cell yield (cells/mg tissue) and tumoroid formation at day 7 post-plating in OncoPro™ Tumoroid Culture Medium.Optimization revealed that initial viable cell yield was not predictive of subsequent culture success, necessitating dual metrics of viability and tumoroid growth. Collagenases, DNase, dispase, and hyaluronidase were beneficial depending on tissue type. While higher yields were obtained in some dissociation conditions, tumoroid formation was compromised, demonstrating a trade-off between digestion of tissue and stress on cells. Approximately 90% of dissociated samples generated tumoroids within 7 days, demonstrating both speed and reproducibility of the optimized workflow. Next-generation sequencing of paired solid tissue, dissociated cells, and day 7 tumoroids revealed >85% correlation in cancer-related gene expression and >95% overlap in single nucleotide variants, confirming preservation of tumor identity throughout processing. We further explored whether long-term tumoroid derivation success could be improved by spike-in of additional supplements and growth factors to OncoPro medium and identified several candidates that potentially contribute to line establishment. This integrated workflow of tumor transport, dissociation, and culture led to optimal viable cell recovery and tumoroid formation efficiency across multiple cancer indications. These results establish a practical and scalable framework for generating high-quality patient-derived tumoroids.