Pancreatic organoid systems have recently been described for the in vitro culture of pancreatic ductal cells from mouse and human. Mouse pancreatic organoids exhibit unlimited expansion potential, while previously reported human pancreas organoid (hPO) cultures do not expand efficiently long-term in a chemically defined, serum-free medium. The aim of this project was to generate a 3D culture system for long-term expansion of human pancreas ductal cells as hPOs to serve as the basis for studies of human pancreas ductal epithelium, exocrine pancreatic diseases and the future development of a genomically stable replacement cell therapy for diabetes mellitus. hPOs can be generated and expanded in a chemically defined, serum-free, human pancreas organoid culture medium with high efficiency from both fresh and cryopreserved primary tissue. Crucially, the hPO culture system also supports the establishment and expansion of these organoids in a chemically defined, modifiable and scalable, biomimetic hydrogel thus facilitating their translation into the clinic. Moreover, hPOs expanded over months in culture maintain their ductal morphology and biomarker expression of the primary tissue while they can also be expanded from tissue with underlying disease such as type 2 diabetes (T2D). This project further demonstrates that hPOs maintain stable chromosomal numbers following long-term in vitro culture, especially when compared to an established positive tumour organoid control. When clonal hPOs were subjected to whole genome sequencing (WGS), they maintain genomic integrity following culture and acquire less mutations in culture than iPSC-derived cultures, resembling other reported organoid systems. Xenografts of hPOs survive long-term in vivo when transplanted into the pancreas of immunodeficient mice. Notably, mouse orthotopic transplants show no signs of tumorigenicity. To further assess their clinical applicability, hPOs were assessed for the expression of antigenic molecules, demonstrating that under in vitro conditions that mimic the inflammatory milieu, hPOs can upregulate HLA Class II. Moreover, this project utilises recently established human immune system (HIS) mice to interrogate in vivo hPO immune rejection under autologous and allogeneic conditions. Lastly, preliminary experiments show that hPOs can be genetically manipulated to express GFP and Luciferase which can be used for in vivo survival and immune rejection tracking.