O180: An In Vitro Cell Model to Assess the Effects of Complement Inhibition on Ischemia-Reperfusion Injury in Cholangiocytes

Abstract

Abstract Background Liver transplantation is the definitive procedure for end-stage liver failure but is limited by a shortage of viable donor organs. IRI is an unavoidable consequence of transplantation, triggering damage via several pathways, including complement activation. Development of an in vitro cell model of IRI with addition of complement allows analysis of cholangiocyte cellular stress response and whether this response can be attenuated by eculizumab, a complement inhibitor. Methods H69 cholangiocytes were subject to anoxic incubation for 4 hours and subsequent normoxia for 24 hours, before addition of complement to the system. Complement was provided from perfusate taken from liver normothermic machine perfusion (NMP). Analysis of cellular response was then carried out at different stages of simulated IRI. Results Eculizumab-treated perfusate significantly increased cell proliferation compared to untreated perfusate (p<0.0001). Addition of complement to the model increased cellular oxidative stress and inflammation. Eculizumab-treated perfusate significantly decreased production of GDF15 (p<0.0001), an oxidative stress marker, in cholangiocytes. Despite a significant increase, compared to the normoxic control, IL-10 and IL-8 production showed no significant difference between cells treated with control NMP perfusate or eculizumab-treated perfusate. VEGF gene expression was upregulated in the context of eculizumab-treated perfusate. Conclusion The in vitro model successfully simulates IRI and addition of perfusate leads to increased production of markers of inflammation and oxidative stress. Complement-depleted perfusate significantly reduced GDF15 production, suggesting amelioration of cellular oxidative stress, by eculizumab; however, we cannot conclude with the current data that complement-depleted perfusate attenuates the effects of IRI in this model.