Introduction: Islet allotransplantation is a possible treatment for Type 1 diabetes. However, immediately following transplantation, islets face acute hypoxic stress due to the lack of vascularization of the newly transplanted tissue. Up to 60% of newly-transplanted islets perish during the first 48 hours post-transplantation as a result of hypoxic injury. In order for islet transplantation to be effective, this period of hypoxia needs to be alleviated. Calcium peroxide (CP) has been reported to be able to increase dissolved oxygen levels in media during in vitro culture. However, it releases oxygen in rapid bursts, damaging islets through the generation of reactive oxygen species. The aim of this study was to examine strategies aimed at eliminating the oxidative burst and achieving a slow, steady release of oxygen using phosphate-complexed calcium peroxide encapsulated within alginate microcapsules.
Materials and Methods: Supplemented RPMI media (Gibco) was deoxygenated after incubation at 0% O2 overnight in a hypoxia chamber (XVivo, Biospherix). Dissolved oxygen levels in the media were confirmed using a dissolved oxygen meter (Oakton D110). Phosphate-complexed calcium peroxide (PcCP) was synthesized by mixing calcium peroxide with phosphate-buffered saline at 10mg/mL and incubating at 37°C overnight. CP and PcCP were encapsulated (eCP & ePcCP) with within microcapsules made with 3 % sodium alginate (Sigma-Aldrich) using an air-driven electrostatic generator (Nisco Engineering AG, 9kV, 3-4psi, 120mM CaCl2). The eCP and ePcCP were supplemented to deoxygenated media containing 100 hand-picked islets isolated from Sprague-Dawley rats using standard protocols. The islets were allowed to incubate with the eCP and ePcCP for a 24 hour period in a controlled hypoxic chamber at 37oC, 5% CO2 and 0% O2. Appropriate controls were used. Islet viability and function analysis studies were performed using standard membrane integrity assays and glucose stimulated insulin release assays respectively. The results were reported as Mean±SEM. Data obtained was analyzed using one way ANOVA followed by a post-hoc Tukey HSD test; p<0.05 was considered statistically significant.
Results: Encapsulated CP & PcCP demonstrated significantly higher dissolved oxygen levels (19.6±3%; eCP, 16.7±1%, ePcCP), that approach normoxic conditions (20% O2). Controls (0.2±0.1%; media, 0.2±0.1%) did not show any significant change (p=0.7). Exposure to hypoxia drastically reduced islet viability (78±5%, 20%O2; 31±1%, 0%O2, p<0.01, ANOVA) and glucose stimulated insulin release (SI: 2.0±0.6, 20% O2, SI: 0.6±0.06, 0% O2, p<0.01, ANOVA). In the treatment groups, after 24 hours of hypoxia, islet viability was preserved (72±2%, eCP; 75±2%, ePcCP, p<0.001, ANOVA); Insulin release was also significantly higher (SI: 1.36±0.3, eCP, SI: 0.8±0.04, ePcCP, p<0.01, ANOVA).
Conclusion: Supplementation of calcium peroxide is a promising strategy to combat low dissolved oxygen levels during prolonged in vitro cell culture and tissue hypoxia during the immediate post-transplant period. In this study, we found that complexing oxygen-generating particles with phosphate and encapsulating within alginate microcapsules reduces the oxidative burst, but preserves sustained oxygen release at concentrations that would be beneficial to islets during in vitro culture or during the immediate post-transplant period.