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BACKGROUND Ex vivo lung perfusion (EVLP) has the potential to increase the number of donor lungs available for lung transplantation (LTx). While the current maximum cold ischemia time (CIT) for donor lungs in clinical LTx is around 8 hours, there is no data regarding the potential use of rejected donor lungs with CIT greater than 8 hours prior to EVLP. The purpose of this study was to investigate the transplant suitability of lungs with a prolonged CIT in two EVLP systems. METHODS Following prolonged CIT of 13.8 hours (range 9.0-19.5 hours), sixteen rejected human donor lungs were randomly divided and perfused using either low flow acellular or high flow cellular EVLP systems (n = 8, each). Transplant suitability was evaluated according to the standard criteria of each EVLP system. RESULTS The high flow cellular group was associated with significantly lower transplant suitability (0% vs. 37%, P = 0.02), significantly lower wet/dry ratio change (-0.71 ± 0.62 vs. 0.43 ± 1.01, P = 0.03) and lower pathological score (1.62 ± 0.61 vs. 3.00 ± 0.61, P = 0.13) than the low flow acellular group. In both systems, inflammatory cytokines on perfusate (TNF-α, IL-1ß, IL-6, IL-8 and IL-10) increased in a time-dependent fashion and were significantly higher than those of controls with CIT less than 8 hours (P < 0.05). CONCLUSIONS The potential for reconditioning lungs with a CIT greater than 8 hours is diminished compared with lungs having a shorter CIT due to severe ischemia reperfusion injury.