Daniel Brandhorst,1,2 Limor Baruch,3 Heide Brandhorst,1,2 Stasia Krishtul,3 Marcelle Machluf,3 Paul R.V. Johnson1,2
1Research Group for Islet Transplantation, Nuffield Department of Surgical Sciences, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom
2Oxford Consortium for Islet Transplantation, Oxford Centre for Diabetes, Endocrinology, and Metabolism (OCDEM), Churchill Hospital, University of Oxford, Oxford, United Kingdom
3Laboratory for Cancer Drug Delivery and Cell Based Technologies, Faculty of Biotechnology and Food Engineering, Technion, Israel Institute of Technology, Haifa, Israel
Aim
Islet isolation essentially requires dissociation of islet basement membranes by collagenolytic enzymes. This is associated with reduced islet function and increased cell death. Previous ex-vivo and in-vivo studies demonstrated that individual extracellular matrix proteins (ECMPs) 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). In contrast to previous studies, ECMPs were dissolved in media rather than coating of culture vessel surfaces.
Methods
Islets, isolated from pancreases of 6 human DBD donors (52±3 years, 28.5±1.5 BMI, 6.4±0.7 hours CIT), were cultured for 4–5 days in hypoxic atmosphere (2% oxygen). Islets were suspended in CMRL 1066 supplemented with 2% FCS and WPM-gel (200 µg/mL) extracted and purified from porcine pancreases. The WPM-gel was compared with a pre-tested combination of human ECMPs composed of 80 µg/mL collagen-IV, 10 µg/mL laminin-521, and 10 µg/mL nidogen-1. Sham-treated islets (STIs) cultured without ECMPs or WPM-gel served as controls. Post-culture characterisation included IEQ yield or islet number (IN), viability (FDA-PI), early plus late apoptosis (annexin V-PI), glucose stimulation index (SI: 2 vs 20 vs 2 mM) and reactive oxygen species production. All parameters were normalised to IEQ, related to pre-culture data if appropriate and presented as mean ± SEM. Statistical analysis was performed by Friedman test and Dunn’s multiple comparison.
Results
Post-culture recovery was highest when hypoxic human islets were cultured in WPM-gel and compared with STIs (65±10% vs 38±10%, p<0.01). Although fragmentation (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). Pre-culture viability was nearly completely preserved when human ECMPs (99±10% vs 79±10%, p<0.01) or WPM-gel (92±8%, p<0.05) were administered. Reactive oxygen species production in STIs increased nearly three-fold (127±15 AU/IEQ) but was halved in the presence of ECMPs (61±14 AU/IEQ, p<0.01) or WPM-gel (65±18 AU/IEQ, p<0.05). While initial early apoptosis remained stable when human islets were treated with human ECMPs (90±13% vs 136±14%, p<0.01) or WPM-gel (84±10%, p<0.01), apo-necrosis increased substantially in the presence of human ECMPs (172±33%, NS) or WPM-gel (154±195 vs 214±24%, p<0.05). Glucose-stimulated islets did not respond adequately after sham-treatment (SI 0.85±0.14). In contrast, supplementation with human ECMPs (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 increased by human ECMPs (56±8% vs 34±8%, p<0.01) or WPM-gel (58±8%, p<0.01).
Conclusion
This initial study presents a new approach to protect human islets from hypoxia-induced damage by supplementing culture media with selected ECMPs or with the whole pancreatic ECM. We assume, that the outcome of our approach will be further improved when the ECM is extracted from human pancreases. These promising findings can be used to develop advanced culture media and innovative encapsulation techniques to protect transplanted islets.
Proteomic Profiling Reveals the Ambivalent Character of the Mesenchymal Stem Cell Secretome: Assessing the Effect of Preconditioned Media on Isolated Human Islets