Preservation of the Endothelial Barrier Function; from Hypothermia to Normothermia Using Different Solutions

Abstract

Introduction: Cold storage of non-heartbeating donor organs can lead to endothelial activation and loss of barrier function, potentially resulting in graft failure. While (sub)normothermia might ameliorate hypothermiainduced endothelial cell damage during preservation, it demands extensive metabolic support. With the advent of novel preservation solutions, our aim was to identify an optimal preservation temperature with matching solution to maintain endothelial barrier function and viability under hypothermic and normothermic conditions. Methods: Human umbilical vein endothelial cells (HUVECs, passage 3) were exposed for 20 hours to 4, 15, 20, 28, or 37°C with a randomly assigned preservation solution comprising: endothelial cell growth medium (ECGM), ringers lactate (RL), university of Wisconsin solution (UW), histidine-tryptophan-ketoglutarate (HTK), or Polysol (PS) that, in contrast to UW, is an extracellular-type solution containing polyethylene glycol. Endothelial barrier function was assessed using a continuous impedance sensing technique (ECIS) of which t=0 was corrected for the pre-experimental impedance and for fluid conductivity. Mitochondrial activity (WST-assay), apoptosis (FLICA-assay, caspase 3&6), and necrosis (Hoechst stain) were assessed on the same cell cultures. ECIS cultures were additionally assessed with flow cytometric determination of percentage inflammation (ICAM-1 and E-selectin), apoptosis (Annexin-V), and necrosis (ToPro-3) in 20k events. Results: A temperature-dependent effect was observed for all solutions on endothelial barrier integrity. At 4°C, HTK, UW, and PS performed equally but, as temperature increased to 20°C, only UW maintained endothelial barrier function (P< 0.01). Above 20°C, only ECGM could maintain endothelial barrier function. Interestingly, UW and RL performed poorly at 20°C regarding mitochondrial activity while HTK and ECGM performed equally well (P< 0.05). Apoptosis was minimal for HTK, PS, UW, and ECGM up to 28°C, but at 37°C, apoptosis was absent only in HUVECs maintained in ECGM (P< 0.05). RL induced considerable necrosis at all temperatures while for HTK, PS, UW, and ECGM a clear turnover-point was identified at 20°C. Above 20°C, only ECGM did not induce necrosis in comparison to the other solutions (P< 0.001). ICAM and E-selectin expression after 20 hours was higher for HTK at 20°C in comparison to RL, PS, and ECGM (P< 0.01) while at higher temperatures no differences were found. Conclusion: At 4°C all solutions stabilized the endothelial monolayer whereas optimal preservation of the endothelial barrier function was observed at 20°C with UW, despite of poor mitochondrial function and inflammation. It appeared that 20°C is a metabolic turnover point after which all assessed preservation solutions could no longer preserve a proper endothelial monolayer or prevent cell death. Figure: The temperature-dependent effect upon endothelial barrier function after 20 hours of preservation.Figure: [Endothelial barrier function]