Vitrification-based tissue-to-organoid cryopreservation workflow for colorectal cancer.

Patient-derived organoids (PDOs) have emerged as powerful preclinical models for capturing the biological and therapeutic complexities of human tumors. However, routine establishment of PDOs from every surgical specimen is time-consuming, costly, and often unnecessary, as actionable cases are identified only after histopathological or genomic analysis. In this study, we developed and evaluated a vitrification-based tissue cryopreservation method that enables on-demand PDO generation from colorectal cancer (CRC) specimens, addressing practical limitations of conventional workflows, including labor-intensive processing and limited flexibility for on-demand PDO generation. Tumor tissues were cryopreserved using multiple vitrification formulations, including conditions containing antifreeze protein (AFP) type III, and evaluated for organoid recovery after thawing. Among the tested conditions, one formulation composed of ethylene glycol, dimethyl sulfoxide, sucrose, and AFP type III (CS#2) showed the most consistent organoid recovery and supported preservation of post-thaw growth, epithelial morphology, and early expansion behavior similar to those observed in matched fresh PDOs. In a matched PDO model, organoids derived from cryopreserved tissue showed drug-response patterns broadly comparable to those of fresh PDOs, providing evidence that functional responsiveness may be retained after tissue cryopreservation. Collectively, these findings support the feasibility of a practical tissue-to-organoid biobanking workflow for CRC that enables flexible, time-independent PDO generation and could facilitate broader implementation of PDO-based functional studies in translational research workflows.