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

Abstract

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.