Superiority of mild hypothermic (20 degrees C) preservation for pancreatic microvasculature using the two-layer storage method.

Hypothermia causes vascular endothelial damage that leads to graft microcirculation disorder and eventually thrombosis after reperfusion. The two-layer cold storage method (TL) was previously demonstrated to supply oxygen to the pancreas graft and maintain high adenosine triphosphate tissue concentration. In this study, we evaluated whether mild hypothermic (20 degrees C) preservation using the TL method could reduce endothelial damage while maintaining parenchymal viability. Graft survival by 20 degrees C preservation was investigated using a dog segmental pancreas autotransplantation model (simple storage in University of Wisconsin solution (UW) for 5 and 8 hours or TL for 5, 8, 12, and 24 hrs. respectively). Subsequently, the grafts were preserved in four different conditions (4 and 20 degrees C UW. 4 and 20 degrees C TL) for 8 hours to evaluate microvascular endothelial damage. Trypan blue uptake of vascular endothelium and pancreatic tissue perfusion were evaluated. No graft preserved by 20 degrees C UW for 5 and 8 hours survived (0/7 and 0/4). In contrast, the graft survival rates by 20 degrees C TL for 5, 8, 12, and 24 hours were 100% (5/5), 80% (4/5), 20% (1/5), and 0% (0/4), respectively. In trypan blue uptake analysis, there were significant differences between 4 and 20 degrees C in both UW and TL (4 degrees C UW, 37% [n = 5) vs. 20 degrees C UW, 13% [n = 4] [p < 0.01]; 4 degrees C TL, 29% [n = 5] vs. 20 degrees C TL, 10% [n = 5] [p < 0.011). The perfusion values in 20 degrees C TL were significantly higher than those in other groups at least for up to 120 minutes after reperfusion (p < 0.01 ). In short-term pancreas preservation, mild hypothermic TL reduced vascular endothelial cell damage and ameliorated graft microcirculation while maintaining parenchymal viability. Mild hypothermic TL may lessen vascular complications in clinical pancreas transplantation when used for several-hour preservation.

The effect of the two-layer cold storage method on islet isolation from ischemically damaged pancreas.

Previous studies have shown that the two-layer cold storage method (TL) resuscitates the function of warm ischemically (WI) damaged pancreas when transplanted as a segmental graft in a dog model. In this study, we evaluated the effect of TL on yield and viability of islets isolated from WI damaged pancreas. In group 1 (n = 10), dog pancreases were processed into islets immediately after total pancreatectomy. In group 2 (n = 5), excised pancreases were left in the abdominal cavity for 60 minutes as WI then processed. In group 3 (n = 5), WI damaged pancreases were preserved in TL at 4 degrees C for 24 hours before islet isolation. Islets were isolated by the digestion methods described by Ricordi, et al. And purified on Euro-Ficoll discontinuous gradients. We compared three groups in terms of islet yields before and after purification, expressed as islet equivalents (IEQ, diameter standardizing to 150 microns) per pancreas weight (IEQ/g pancreas). Viability of islets was assessed with in vitro static incubation test. Islet yields after purification were 2500 +/- 1100 (mean +/- SD) in group 1, 1200 +/- 800 in group 2, (p = 0.04 vs. group 1, p = 0.03 vs. group 3), and 2200 +/- 300 in group 3 (p = 0.38 vs. group 1). Static incubation test revealed that islets in group 1 and 3 showed sufficient insulin release in response to glucose stimuli. These data clearly demonstrated that TL can regain islet yield from WI damaged pancreas up to similar level to that from fresh pancreas. Viability of those islets was well maintained. Thus temporary TL applied before islet isolation may allow to use islets from non-heartbeating cadavers in the clinical setting.