CELL PRESERVATION IN UNIVERSITY-OF-WISCONSIN SOLUTION DURING ISOLATION OF CANINE ISLETS OF LANGERHANS

Allogeneic islet transplantation in Type I diabetic patients is consid erably hampered by the variable outcome of islet isolation and purific ation. After collagenase digestion of the pancreas, islet isolation is traditionally performed under hypothermic conditions in physiological solutions such as Hanks and RPMI. The University of Wisconsin solutio n (UWS) has been shown superior for hypothermic preservation of the pa ncreas. We, therefore, compared the UWS and RPMI for canine islet isol ation and subsequent purification in either a conventional hyperosmoti c density gradient of dextran in Hanks, or a novel normosmotic density gradient of Percoll in UWS. The isolation solution did not affect isl et yield before purification (51% of the native islet mass). Loss of a mylase (30%) and swelling of the acinar cells were observed in RPMI. I n contrast, no loss of amylase and slight shrinkage of the acinar tell s were observed in the UWS. Cell swelling affected the density separat ion and viability of the cells. Dextran density separation resulted in a 15% purity and 41% recovery of the islets isolated in RPMI, as comp ared to a 93% purity and 52% recovery of islets isolated in UWS. Perco ll density separation improved the purity (99%) and recovery (74%) of islets isolated in UWS. Islets isolated in UWS demonstrated a superior basal and glucose stimulated insulin release during perifusion. Elect ron microscopy demonstrated a well-preserved islet ultrastructure afte r isolation in both solutions - except for slightly swollen mitochondr ia after isolation in RPMI. Autotransplantation of islets in pancreate ctomised dogs was successful both after isolation in UWS and RPMI. We conclude that prevention of cell swelling during isolation and purific ation in the UWS resulted in an improved yield of viable and consisten t virtually pure islets. Prevention of cell swelling during islet isol ation should facilitate the analysis and control of other factors affe cting outcome in man.