Whole Porcine Pancreatic Extracellular Matrix-Based Hydrogel Increases Survival Of Human Islets Exposed To Hypoxia

Aim: Islet isolation essentially requires the dissociation of the islet basement membrane by collagenolytic enzymes. Basement membrane loss is associated with reduced islet function and increased cell death. Previous ex-vivo and in-vivo studies demonstrated that individual extracellular matrix (ECM)-proteins can increase islet survival. As the natural ECM is a tissue-defined complex network we propose a novel concept for creating a specific islet matrix by using the whole pancreatic ECM (WPM). Methods: Islets, isolated from 6 human pancreases, were cultured for 4–5 days in 2% oxygen. Islets were suspended in CMRL 1066 (2% FCS) and WPM-gel (200 µg/mL) extracted and purified from porcine pancreatic tissue. The WPM-gel was compared with a pretested combination of 80 µg/mL human Collagen-IV, 10 µg/mL Laminin-521, and 10 µg/mL Nidogen-1 (CLN). Sham-treated islets cultured without any ECM components served as controls. Post-culture characterisation included yield of islet equivalents (IEQ) or islet particle number (IN), viability (FDA-PI staining), early and late apoptosis (Annexin V-PI), glucose stimulation index (SI: 2 vs 20 vs 2 mM) and production of reactive oxygen species (ROS). All parameters were normalised to IEQ, related to pre-culture data if appropriate and presented as mean ± SEM. Results: Post-culture yield was significantly highest when hypoxic human islets were cultured in WPM-gel and compared to controls (65±10% vs 38±10%, p<0.01). Although fragmentation, determined as IN/IEQ ratio, increased after all treatments, this increase was lowest in the presence of WPM-gel (0.62±0.05 vs 0.80±0.14 vs 0.93±0.27, NS). FDA-PI staining revealed that pre-culture viability was preserved by nearly 100% when CLN (99±10% vs 79±10%, p<0.01) or WPM-gel (92±8%, p<0.05) were administered. ROS production in controls increased nearly 3-fold (127±15 AU/IEQ) but was significantly halved in the presence of CLM (61±14 AU/IEQ, p<0.01) or WPM-gel (65±18 AU/IEQ, p<0.05). Whilst initial early apoptosis remained stable when human islets were treated with CLN (90±13% vs 136±14%, p<0.01) or WPM-gel (84±10%, p<0.01), apo-necrosis increased substantially in the presence of CLN (172±33%, NS) or WPM-gel (154±195 vs 214±24%, p<0.05). Control islets did not respond with adequate insulin secretion after glucose stimulation (SI 0.85±0.14). In contrast, supplementation with CLN (1.29±0.09, p<0.05) or WPM-gel (1.34±0.09, p<0.01) preserved the physiological insulin response during hypoxia. Overall survival, considering the recovery of viable cells only, was significantly increased using CLN (56±8% vs 34±8%, p<0.01) or WPM-gel (58±8%, p<0.01). Conclusion: This initial study presents a novel and efficient approach to protect human islets from hypoxia-induced damage by supplementing culture media with selected ECM-proteins or with whole pancreatic ECM. The promising findings may serve as starting point for a new encapsulation technique to protect transplanted islets. European Union’s Horizon 2020 (645991). Juvenile Diabetes Research Foundation (JDRF) award (31-2008-617).