Isolamento di cellule staminali provenienti dal dotto di Wirsung del maiale e differenziazione in vitro in cellule beta simili

[automatically translated] Introduction: The purpose of this study is to obtain a protocol for the isolation and in vitro culture of stem cells from the pig Wirsung duct and observe the potential of such cells in differentiation into insulin-producing β-like cells. Materials and methods: The Wirsung duct was isolated by microdissection after surgical removal of the pig duodenum-pancreas block by in situ perfusion levy and subsequently digested first with 1 mg / ml of collagenase P and then with 2mg / ml of trypsin to obtain ductal stem cells, which subsequently were placed in culture medium (serum-free) with DMEM without glucose with the addition of growth factors. Subsequently the cells were cultured with increasing concentrations of glucose (0-control, 5.6 mM / L, 17, 8 mM / L and 25 mM / L) and subsequently identified by immunohistochemical staining results: In the first two weeks there has been a very slow cell growth; after induction with glucose at different concentrations for two weeks, it is noted the ability of such cells to aggregate and to build islet-like structures. These cells were stained using the common pancreatic ductal markers (CK19, insulin, glucagon). After the second week, 90% of cells of the pancreatic dimensional structures turned out positive for CK19. In addition 21.5% of these stimulated by glucose in the culture medium tested positive for insulin and less than 3.2% for glucagon. The percentage of immunopositive cells for glucose resulted dependent on the concentration of glucose in the middle; it was observed that the concentration of 17.8 mM / L glucose in the culture medium has effectively stimulated the secretion of insulin-glucagon of these cells. Conclusions: The stem cells from the pig Wirsung duct are capable of in vitro proliferation and have the potential, induced by glucose, to differentiate into insulin-producing β-like cells.