Studies on the isolation and transplantation of mammalian pancreatic islets.

A review is made of the relationship between the disease diabetes mellitus and the insulin-secreting beta cells of the islets of Langerhans. In addition, an account is given of the clinical and experimental methods of treatment including insulin therapy and transplantation of the pancreas both as an intact organ and as isolated endocrine tissue (islet transplantation). In particular the methods which have been used to release and isolate islets from the rodent, large animal and human pancreas are discussed. Furthermore, the metabolic effects of transplantation, results of allogeneic transplantation and techniques of islet preservation are described. In this thesis I have set out to determine whether the standard islet isolation method (using density gradients of Ficoll medium) could be improved by utilising an alternative density gradient medium (Bovine Serum Albumin). Using a rodent model, islet yield, purity and viability (both in vitro & in vivo) were shown to be improved with the BSA method over the standard Ficoll technique. A second stage was subsequently performed, using a novel test gradient system, to optimise the BSA gradient method further by definition of the precise density of rat islets and exocrine tissue. The BSA gradient method was then adapted to the isolation of canine islets. To overcome the difficulties in processing large volumes of digest produced from the collagenase digested pancreas, an IBM 2991 cell separator was adapted to produce large volume density gradients. Although high yields of highly purified and viable islets were obtained, these were insufficient to prevent diabetes ensuing in autotransplanted pancreatectomised dogs. An animal model was developed which would potentially be applicable for the in vivo assessment of human islet viability. The nude (athymic) rat, allogeneic and xenogeneic (mouse) islet transplants and a short diabetic induction period with streptozotocin were all used. Finally, the BSA gradient methodology and IBM 2991 cell processor were used for the large scale purification of intact human islets which were shown to be viable both in vitro, and in vivo following transplantation into diabetic nude rats.