An investigation into the factors influencing the purification of mammalian pancreatic islets for transplantation.

The treatment of Type 1 (Insulin-Dependent) Diabetes Mellitus by the transplantation of isolated islets of Langerhans is an attractive concept, but one which has been difficult to realise in clinical practice. Insulin-independence has been relatively rare following human islet allotransplantation, due partly to immunological rejection of islets, but also due largely to the problems inherent in the isolation of sufficient numbers of purified islets from the native pancreas. The purification of islets prior to transplantation is desirable for several reasons, and is usually achieved using large-scale density gradients. Current techniques of density-dependent separation of islets are inefficient, however, and result in an unacceptable loss of islet yield. The purpose of the work described in this thesis was therefore to examine the possible factors limiting the efficiency of islet purification using density gradients, and to investigate ways in which these factors may be usefully modified. Using a standardised, quantitative system for assessing the efficiency of islet purification on density gradients, it was demonstrated that the separation of purified human and porcine islets was highly dependent upon the physico-chemical environment of pancreatic tissues throughout the isolation process. In particular, it was shown that islet purity is compromised by exocrine tissue swelling occurring during the isolation procedure, but that this swelling can be reversed, and islet purity thereby markedly improved, by suspension of the dispersed, collagenase-digested pancreatic tissues in appropriate storage solutions. The optimal, composition of such solutions was defined, and their combined use both for storage of the dispersed pancreas and as a solvent for novel density gradient media was examined. The results obtained in this way have contributed to an improved understanding of the factors potentially influencing islet purification, and may therefore assist in expediting the clinical application of islet transplantation as a therapy for diabetes