ISOLATION OF PORCINE PANCREATIC-ISLETS FOR XENOTRANSPLANTATION STUDIES - EFFECTS OF LOW COLLAGENASE DIGESTION TEMPERATURES

Islets of Langerhans were isolated from porcine pancreata by a modific ation of our previously described method. The modification involved th e use of a low temperature of collagenase digestion (30 degrees C) dur ing the process of islet isolation. The resulting islets were then eva luated in vitro and in vivo and compared to islets isolated at the reg ular 37 degrees C temperature. The islets produced at the low temperat ure were more compact compared to the control islets. In the dextran d ensity gradient these islets were deposited at the interface of the 1. 060 and 1.068 g/ml density bands as compared to 1.050 and 1.060 g/ml f or the control islets. In addition, the experimental islets contained a higher proportion of compact, unfragmented islets (68%) compared to the regular islets (55%), and their uptake of the dithizone stain was considerably slower than with the control islets. All ten batches of f reshly isolated microencapsulated islets produced at both temperatures responded to the glucose stimulation. After 4 weeks of in vitro cultu re the islets of both groups microencapsulated in alginate-polylysine- alginate (APA) microcapsules still retained glucose responsiveness, wi th the experimental islets demonstrating significantly higher responsi veness to the high glucose (16.7 mM) and 0.1 mM IBMX stimulation. The morphology of unencapsulated islets in the experimental group followin g 4 weeks of in vitro culture indicates much firmer islet structure co mpared to the control islets. In addition, the unencapsulated experime ntal islets following the 4 week culture were still found to have secr eted insulin when exposed to glucose. In transplantation studies both the experimental and the control islets normalized diabetic hyperglyce mia in diabetic mice in a comparable fashion. In general, the low temp erature digestion results in superior islets in terms of their morphol ogy, viability, and physiological function.