Improved quality and yield of islets isolated from human pancreata using atwo-step digestion method

A new approach, involving a two-step digestion process and Los Angeles pres ervation solution #1 (LAP-1), a cold storage solution, was developed for is olation of high-quality islets from human pancreata for transplantation. Th is approach markedly improves the islet yield, purity and viability, and th e isolation success rate. In this method, the pancreas was digested first i n warm collagenase solution for up to 20 minutes. After decanting the enzym e solution, partially digested tissue was dissociated by gentle agitation i n cold LAP-1 solution without additional collagenase. The digested tissues were stored in cold LAP-1 solution until islet purification on Euro-Ficoll. Forty-six islet isolations were performed consecutively by the new method (group 1). These results were compared to those obtained earlier with 46 co nsecutive isolations, using our previous method that had been used before d evelopment of the new method (group 2). Our old method was a modification o f Ricordi's method involving only warm collagenase digestion and the storag e of digested tissues in cold Hanks balanced salt solution. All pancreata w ere partial, containing the body and rail. Then were no significant differe nces in both groups with regard to the donor age, cold ischemic time, harve sting conditions, and pancreatic weight. Pancreas digestion was completed i n approximately 1 hour in both groups. The isolation success rate as determ ined by viable islets after 2 days in culture was 93.5% (43 of 36 cases) in group 1, and 56.5% (26 of the 46) in group 2. immediately after isolation, the new method yielded a total of 335,739 +/- 36,234 islets equivalent to 150 mu m (IEQ) and 6,233 +/- 681 IEQ/g of pancreas with 83 +/- 2.5% purity, whereas the old method yielded a total of 195,587 +/- 25,242 IEQ and 3,763 +/- 5,509 IEQ/g with 69.2 +/- 4.7% purity. Isolated islets in group I main tained a good three-dimensional structure, displayed normal insulin release to high glucose stimulation in vitro, and restored euglycemia after transp lantation into streptozotocin-diabetic athymic mice. The two-step digestion method provides a sufficient number of islets for transplantation from a s ingle pancreas.