Isolation and colture of beta-like cells from porcine Wirsung duct

Abstract

We sought to develop a protocol to isolate and culture porcine Wirsung duct cells in order to determine their potency to differentiate into insulin-expressing beta-like cells. The porcine Wirsung duct isolated by a surgical microdissection was digested with collagenase P and trypsin to dissociate ductal cells. These elements were cultured in serum-free supplemented media: for 2 weeks. Thereafter the cells were exposed to varying concentrations of glucose (0, 5.6, 17.8, and 25 mmol/L) to induce a beta-like phenotype, as identified by immunohistochemical staining. Cell growth proceeded slowly for the first 2 weeks of culture. After glucose induction for 2 weeks, they formed pancreatic islet-like structures. These cells were stained for the pancreatic ductal cell marker cytokeratin-19 (CK-19) and the pancreatic endocrine markers insulin and glucagon. After the second week, 90% of cells were positive for CK-19. Up to 20.1% of the cells in pancreatic 3-dimensional structures induced by 17.8 mmol/L glucose were positive for insulin, and <3.2%, for glucagon. The positive ratio of immunoreactive staining was dependent on the glucose concentration; 17.8 mmol/L glucose effectively stimulated insulin- and glucagon-secreting cells. We concluded that porcine Wirsung duct cells were capable of proliferation with the potential to differentiate toward beta cells upon glucose induction in vitro.