Protocol for generation and utilization of patient-derived organoids from multimodal specimen

Patient-derived organoids (PDOs) are robust preclinical models for precision oncology. However, most existing protocols depend on surgically resected specimens, limiting their applicability to patients who are ineligible for surgery. Here, we present a protocol for generating PDOs from various clinically accessible specimens—including biopsies (endoscopic ultrasound-guided fine needle biopsy EUS-FNB, percutaneous liver biopsy PLB, ascites, and pleural effusion)—across cancer types. We describe steps for specimen transport, tumor cell isolation, culture, biobanking, and high-throughput drug screening, supporting reproducible PDO applications in translational research across diverse clinical settings. For complete details on the use and execution of this protocol, please refer to Lee et al. 1 • Protocol for PDO establishment across cancer types • Standardized workflows for PDO generation, cryopreservation, and drug screening • Applicable to surgical and biopsy samples, including EUS-FNB, PLB, and body fluids • Guidelines to overcome practical challenges in organoid establishment Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Patient-derived organoids (PDOs) are robust preclinical models for precision oncology. However, most existing protocols depend on surgically resected specimens, limiting their applicability to patients who are ineligible for surgery. Here, we present a protocol for generating PDOs from various clinically accessible specimens—including biopsies (endoscopic ultrasound-guided fine needle biopsy EUS-FNB, percutaneous liver biopsy PLB, ascites, and pleural effusion)—across cancer types. We describe steps for specimen transport, tumor cell isolation, culture, biobanking, and high-throughput drug screening, supporting reproducible PDO applications in translational research across diverse clinical settings.