Cryopreservation of islets of Langerhans: Optimization of protocols using rat pancreatic tissue

Abstract

SUMMARY Banking of pancreatic islets following cryopreservation has been shown to be the most effective method facilitating sufficient availability for transplantation to reverse type 1 diabetes. As success of transplantation depends on number of functioning islets the aim of this study was to determine crucial variables of cryopreservation to increase postthaw viability. Isolated rat pancreatic islets were cryopreserved using a protocol systematically changed in several variables: (i) freezing media (RPMI-1640 + 10% FCS, 100% FCS, hepatocyte suspension buffer); (ii) final concentration of cryoprotectant (Me2SO 5%, 10%, 14.4%, 18.3%, 21.7%); (iii) conditions of equilibration (15 min vs 45 min, 22°C vs on ice); (iv) postthaw removal of cryoprotectant (15 vs 30 min; stepwise vs one-step). Concerning freezing media RPMI showed highest viability (60.7±1.5% vs FCS 45.3±1.8%, p<0.05 and hepatocyte suspension buffer 56.3±1.4%, ns). Optimal Me2SO concentration revealed to be 14.4% resulting in 60.5±1.0% viability compared to 22.0±0.9% with 5% Me2SO (p<0.05), 49.5±1.3% with 10% (p<0.05), 19.6±1.3% with 18.3% (p<0.05) and 8.6±0.8% with 21.7% (p<0.05). An equilibration with cryoprotectant on ice showed significantly higher viability than at 22°C (66.7±1.1% vs 60.7±1.1%; p<0.05), whereas more rapid equlibration within 15 min instead of 45 min showed no improvement. For postthaw removal of cryoprotectant stepwise dilution within 30 min (68.9±1.1%; 15 min: 68.7±1.2%) was found to be superior to a one-step technique (65.6±1.2%; p<0.05). Our study determined freezing media, cryoprotectant concentration, conditions of equilibration and postthaw removal of cryoprotectant as important parameters giving the opportunity to further advances of low temperature banking of isolated islets.