Organoid culture is a novel in vitro culture method that promotes the growth of primary and PSC-derived cells in three-dimensional culture to generate structures that recapitulate tissue morphology. We have developed PancreaCult™ Organoid Medium Kits (Human), which combine PancreaCult™ Organoid Initiation Medium (OIM) and serum-free PancreaCult™ Organoid Growth Medium (OGM) into a robust and standardized workflow for the establishment and expansion of pancreatic duct organoids. To establish organoids, fresh or cryopreserved islet-depleted exocrine tissue was seeded in Corning® Matrigel® domes and cultured in PancreaCult™ OIM. After 3 days, the medium was changed to PancreaCult™ OGM and the organoids were passaged and maintained in this medium thereafter, and passaged as fragments every 4 - 7 days. Organoids were successfully established from all 5 donors with a 2- to 6-fold expansion in cell numbers every 7 - 14 days. Real-time PCR and immunocytochemistry demonstrated that these organoids express pancreatic duct markers (PDX1, SOX9, KRT19, CFTR, CA2, and MUC1), as well as proliferation and stem cell markers, Ki-67 and LGR5, respectively. Pancreatic duct organoids could be maintained for at least 10 passages (n = 3) and cryopreserved as fragments that could be used for rapid re-establishment of cultures at later time points. Organoids could also be dissociated and re-seeded in Prostaglandin E2-free PancreaCult™ OGM supplemented with ROCK inhibitor, Y-27632 and 10% FBS on Transwell® inserts to create 2D monolayers. In addition to normal pancreatic duct cultures, PancreaCult™ OGM supported the efficient long-term expansion of 4 pre-established pancreatic ductal adenocarcinoma (PDAC) organoid lines and maintained the mutational profile of the parent line after 5 passages, as determined through whole exome sequencing of 29 cancer-associated genes. Through the optional removal of epidermal growth factor (EGF), PancreaCult™ OGM can be modified to suppress normal cell growth to select for tumor cells with activating KRAS mutations. Our results demonstrate that PancreaCult™ Organoid Medium Kits provide a robust and flexible in vitro culture system for the establishment and expansion of normal pancreatic duct cells, as well as the long-term expansion of PDAC organoids. Citation Format: Nina Quiskamp, Angela Tsai, Joanna Pan, Misha Hasan, George Chang, Isabella T. Tai, Allen C. Eaves, Sharon A. Louis, Ryan K. Conder. PancreaCult™ Organoid Medium Kits support the robust establishment and expansion of human pancreatic duct organoids [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A077.