LONG-TERM PRESERVATION OF THE PIG PANCREAS BY THE ONE-LAYER METHOD FOR SUCCESSFUL ISLET ISOLATION

Abstract

P33 Aims: The oxygenation of the human pancreas during cold storage was recently established for clinical islet transplantation by introduction of the two-layer method (TLM). Nevertheless, simplification of TLM would facilitate its application as a broadly used pancreas preservation method for subsequent islet transplantation. Preliminary data about vascularized pancreas transplantation in rats and dogs indicate that successful preservation by a one-layer method (OLM) omitting UW solution (UWS) as the second layer is also possible. The purpose of the present study was therefore to examine the efficiency of OLM for pig islet isolation after 7 hours of cold ischemia. Methods: After resection adult pig pancreata were intraductally flushed with 2 mL/g of cold UWS. Subsequently, pancreata were either continuously oxygenated during cold ischemia by the TLM (n=10) or OLM (n=8), or processed promptly (n=6). Immediately before digestion-filtration pancreata were intraductally distended with cold UWS supplemented with Collagenase NB 8 and Neutral Protease NB (SERVA). Isolated islets were isolated and purified as previously described. Quality control included determination of islet yield expressed as islet equivalents (IEQ), isolation index (IEQ/islet number), trypan-blue exclusion, glucose stimulation index during static incubation (20 vs 2.8 mM glucose), insulin and ATP content, and mitochondrial activity measured as formazan production at 490 nm and expressed as optical density (OD). Islet in vivo function was evaluated after transplantation into diabetic nude mice. Results: No significant differences were found regarding islet purity (>90%) and viability (>95%). Compared to freshly procured pancreata (429200±86700 IEQ) postpurification islet yield was significantly decreased (P<0.05) after prolonged cold storage by TLM (238000±26600 IEQ). In contrast, islet yield was significantly higher (P<0.05) in cold-stored organs using OLM (338600±42100 IEQ, NS vs fresh). Metabolic islet integrity was not different between unstored pancreata and organs preserved by OLM or TLM as indicated by the intracellular insulin content (710±130 vs 830±120 vs 750±70 μU/IEQ), formazan production (0.7±0.1 vs 0.6±0.1 vs 0.7±0.1 OD/1000 IEQ) and ATP content (390±70 vs 370±80 vs 410±170 pg/IEQ). In contrast, glucose stimulation index was significantly decreased in islets isolated after TLM-storage (1.8±0.2, P<0.05) but not after OLM-storage (2.3±0.6, NS) compared to freshly isolated islets (2.5±0.4). Transplantation into diabetic nude mice demonstrated islet graft function in all experimental groups. Conclusions: The present study demonstrates clearly that viable pig islets can be successfully isolated from long-term stored pig pancreata utilizing perfluorochemicals as a single layer for oxygenation of pancreatic tissue during cold storage. This methodological simplification of the TLM might be of relevance for broad application in clinical islet transplantation.