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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.

K Rozenberg 1, L Knijff 1, J M Sierra Parraga 2, L Lo Faro 1, M J Hoogduijn 2, C 2, J Hunter 1, R Ploeg 1 Nuffield Department of Surgical Sciences, University of Oxford, Oxford, United Kingdom Department of Internal Medicine, Nephrology and Transplantation, Erasmus Medical Center Rotterdam, Rotterdam, The Netherlands BackgroundDonation after circulatory death kidneys are widely used to increase the donor pool, despite their inferior quality. Normothermic machine perfusion (NMP) could provide a platform to assess organ viability prior to transplantation and offers the unique opportunity for active interventions to an isolated organ. Mesenchymal stromal cells (MSC) have been shown to possess potent anti-inflammatory and regenerative properties ameliorating ischaemia reperfusion injury. However, the most common delivery route of MSC is intravenous infusion, which is associated with off-target distribution. We aimed to determine the dose of MSC needed to allow successful homing in donor kidneys during NMP without adversely affecting renal perfusion dynamics. MethodsPorcine slaughterhouse kidneys with 20 min warm ischaemia were retrieved and underwent 3h hypothermic machine perfusion followed by NMP for 7h. An oxygenated, autologous blood-based solution containing albumin, electrolytes and nutrients was used as perfusate. Following 1h of NMP, either a vehicle, 2 x 106, 10 x 106 or 50 x 106 labelled (Qdots) porcine adipose derived MSC (n=3 per group) were injected into the cannulated renal artery. Physiological recordings were taken regularly. Perfusate, urine and biopsies were obtained for biomarker analysis and to locate MSC. ResultsNo difference was found in average renal blood flow (p=0.668), renal resistance (p=0.828) and urine production (p=0.307) between all MSC groups compared to control. Damage markers LDH and AST increased during NMP and remained similar between groups (p=0.816 and p=0.312). Confocal microscopy demonstrated mainly glomerular localization of MSC (Figure 1), but they were also observed in the capillary network around the tubules. Additionally, a significant percentage of localised MSC was observed in the glomeruli in the 10 and 50 million MSC groups compared to 2 million MSC (p=0.002 and p<0.0001). No differences were observed between the 2 and 10 million MSCs group (p>0.05). There is a significant increase in secreted inflammatory mediator PGE2 with time in all groups (P<0.05). DiscussionAdministration of different doses of MSC to the donor kidneys during NMP is safe and feasible. MSC successfully homed in the glomeruli. This model provides us with the opportunity of getting a better insight in the interaction between MSC and injured donor kidney: how do MSC or their released secretome affect tissue integrity and renal function.

Alexander Cui, Srinivasa Rao, Sam Olechnowicz, James Edwards, Peter Cook, Edmond Walsh, Claire Edwards Prostate cancer (PCa) cells predominantly metastasise to bone, and once this happens the disease is incurable. Hence it is vital to understand the mechanism of bone metastasis in PCa. Several models have been established to study PCa bone metastasis in vitro, involving the co-culture of PCa cell lines with cells of the bone microenvironment such as osteoblasts, osteoclasts, bone marrow stromal cells, etc. However, these models are limited to one-to-one co-culture (i.e., PCa cells co-cultured with one other cell type), which overly simplifies the complex bone microenvironment consisting of multiple cell types. We aimed to overcome this limitation by adapting a microfluidics platform that employs a novel mechanism to isolate sub-microliter volumes using fluid walls. By using this technology, we were able to successfully differentiate osteoblasts and adipocytes from precursor cells (2T3 and ST2 cells respectively) in nanowells, and co-cultured them with PC3 prostate cancer cells. Differentiation to osteoblasts and adipocytes was confirmed by calcein green and BODIPY live staining which could be monitored using the Incucyte imaging platform. Following co-culture, the PCa cells were trypsinised and total RNA was extracted from them. A cDNA amplification step was successfully incorporated into the reverse transcription protocol to provide sufficient material for quantitative PCR analysis. Further, sequencing adapters were added to the cDNA to generate an RNA-sequencing library for transcriptome-wide analysis. Using this novel model, with imaging, RNA-sequencing and qPCR as read-outs, we are working on elucidating the signalling pathways that are activated by the bone microenvironment in PCa cells.