Isolation, culture, and characterization of endocrine cells from 6-month-old porcine pancreas

Porcine endocrine cells were isolated from pancreas of 6-month-old pigs by two-step enzymatic digestion procedures. They were separated by the density gradient (isopycnic) centrifugation method using Histopaque-1077. Isolated cells were cultured and divided into two groups: suspension cells and adhesion cells. Suspension cells maintained their cell numbers on and after 7 days in culture. Approximately 1 x 10(7) cells were obtained from single pancreas of a 6-month-old pig. The cultured suspension cells took up dithizone (DTZ) staining 14 days after isolation in culture and indicated the presence of beta-cells. In in vitro study, the suspension cells were capable of secreting insulin into the culture medium. The suspension cells were tested for insulin and glucagon staining by Western blot analysis. These results indicated the maintenance of endocrine cell function after isolation. However, cultured adhesion cells failed to maintain their function during culture. In in vivo study, the suspension cells were transplanted into diabetes-induced nude mice. Reduction in blood glucose level was obtained after transplantation. Intraperitoneal glucose tolerance test (IPGTT) results showed a normal pattern of blood glucose clearance. After 1 week, the transplanted endocrine cells were detected with anti-insulin antibody by immunostaining and it showed the presence of viable beta-cells under the renal capsule of nude mice. Collectively, our results suggest that isolated and cultured suspension porcine endocrine cells maintained their endocrine function. These endocrine cells can be used as isolated islets for further study, including transplantation experiments.