Spheroid Culture of Human Pancreatic Ductal Cells to Reconstitute Development of Pancreatic Intraepithelial Neoplasia

: Pancreatic ductal adenocarcinoma (PDA) presents poor 5-year survival rate, mainly attributable to late diagnosis due to its asymptomatic nature. Therefore, building human cell-based systems that reconstitute hallmark features of the PDA precursors, pancreatic intraepithelial neoplasia (PanINs), will accelerate development of new strategies for early diagnostics and intervention. We previously demonstrated that systematic introduction of genetic modification (KRAS, CDKN2A, SMAD4, and TP53) leads to immortalization of primary human pancreatic cells and, upon orthotopic transplantation, their development to human PanIN-like lesions. Here, we describe detailed methods for fluorescence-activated cell sorting, lentiviral transduction, and three-dimensional spheroid culture of primary adult human pancreatic ductal cells, as well as a method for clonal selection of human pancreatic ductal spheres.