Manual adult porcine islet isolation technique and optimal condition for adult pig islets

TERU OKITSU
Institute of Industrial Science, The University of Tokyo

In the case of requiring a high islet yield obtained efficiently through a single series of pancreas procurement and islet isolation procedure, adult pigs, especially retired breeders, are considered suitable to the islet donors. Besides the age of the pig, its race and particular feeding protocols could also influence the number and the morphology of the islets in the pancreas. Because islet yield depends on the naïve number and how well morphology is preserved in the isolated islets, a series of pancreas procurement and islet isolation is recognized to be an important factor to determine islet yield. This presentation will introduce a simple islet isolation procedure that is modified based upon the static digestion method originally described by O'Neil et al [2001 Cell Transplantation].

In the procedure, at slaughterhouse, a whole pancreas from a retired breeder pig is procured within 30 min of warm ischemic time. A cannula is inserted into the main pancreatic duct, which had been connected to the duodenum. Through the cannula, 200-300 ml of M-Kyoto solution is infused into the pancreatic duct. Then the pancreas is preserved in oxygenized perfluorocarbon and transported to the laboratory. At the isolation laboratory, pancreas is distended using 300-400 ml of Hank's balanced salt solution containing 0.5 mg/ml of collagenase. The pancreas is cut into seven to nine pieces, put into a one liter Nalgene jar, and left in water bath at 37℃ for approximately one hour until almost all the pancreas is digested. After the serous membrane of the pancreas is teared off using two pairs of forceps to allow the dissociated tissue to be released into the solution, all the tissue is filtrated through 500 μm mesh in a large filtration chamber and collected in 250 ml conical tubes. For islet purification, four 500 ml plastic containers with flat bottom are used to load discontinuous density gradients. This system allows us to purify up to 120 ml of dissociated pancreas tissue at a time. The discontinuous gradients consist of 100 ml each of M-Kyoto solution containing iodixanol at the density of 1.060, 1.096 and 1.110 g/ml; the bottom gradient of 1.110 g/ml solution is mixed with the dissociated pancreas tissue in advance. This system is centrifuged at 1000 rpm (240 x g) for 5 min at 4℃. The purified islets form a layer between the top and the second gradients and they are carefully collected and washed three times.

Using this islet isolation method, we obtained a mean of 560,000 islet equivalent after purification with islet purity of greater than 60% (n=6 isolations) from one adult pig.  This method would be useful to isolate porcine islets of quite a few numbers in a stable and efficient manner through a single series of pancreas procurement and islet isolation using adult pigs.