Pancreas Digestion Increases Hypoxia and Stress Markers During Human Islet Isolation

Abstract

Introduction: The human islet isolation procedure is known to be metabolically stressful for the pancreatic tissue and may be accompanied by hypoxia. The aim of this study was to identify and compare the hypoxia-induced deleterious changes at sequential steps of the isolation process. Methods: With appropriate consent and ethical approval, human pancreases were retrieved from multiorgan donors. 1ml of tissue samples were collected from the islet isolation circuit, during i) collagenase digestion, ii) digest collection, iii) islet purification, and iv) from the final islet preparation (n=18 islet isolations). Measurements of HIF-1α translocation in the nuclear extract (ELISA), superoxide dismutase activity (enzymatic assay), and caspase 3 activation (ELISA) in the cytoplasmic extract were determined. Results from samples taken at different time points were compared with the first sample taken during digestion (t=0; 10 minutes after closing the chamber). Results: HIF-1α translocation into the nucleus was significantly increased (1.5±0.2 vs. 1.0±0.0; p< 0.001) throughout the first 30 minutes of the digestion and collection processes. Surprisingly, the level of HIF1α in the nuclei remained high after digestion even with the temperature reduced to 4°C (1.7±0.4). Superoxide dismutase activity was variable throughout the isolation process, with an increase during digestion (122.7±13.0%), a decrease during collection (41.5±11.3%), and an increase again during purification (131.6±20.0%). Caspase 3 activation was stable during digestion and purification, but was increased in final islet preparations (2.4±0.9 vs. 0.9±0.4 t=0) when the islet yield was low (< 2500 IEQ/g pancreas), but decreased with higher yields (>2500 IEQ/g pancreas), (0.2±0.4 vs. 1.2±1.1 t=0). Conclusion: From an early time point in human islet isolation, hypoxia and oxidative stress may influence the outcome of the islet isolation by activating the apoptosis pathways. By modifying islet isolation solutions with antioxidants and oxygen supply, we may be able to prevent this and improve the outcome of islet isolation.