Isolated liver perfusion offers a unique prospect for safe, effective targeting of gene therapies that can be directed against allograft rejection or recurrent diseases such as reinfection by hepatitis C virus (HCV). We aimed to examine the effect of organ preservation solutions on vector-based gene therapy delivery under hypothermic conditions. University of Wisconsin (UW) solution, histidine tryptophan ketoglutarate (HTK), EloHaes, sodium-poly(ethylene glycol)-UW solution [Institut Georges Lopez 1 solution (IGL-1)], and Dulbecco's modified Eagle's medium (DMEM) culture medium (control) were tested at 2°C or 37°C for lentiviral vector transduction efficiencies to the hepatoma cell line Huh-7 and primary human or mouse hepatocytes. Lentiviral vectors expressing short hairpin RNA were used to target HCV replication. With a potent short hairpin RNA vector, transductions were directly correlated to the therapeutic effect, with low transduction yielding low knockdown and vice versa. Green fluorescent protein (GFP) reporter gene expression was observed with vector incubation times as short as 10 minutes. The highest transductions were seen, after 2-hour 37°C incubation, in UW (62% ± 6 SEM); they were significantly higher than those in HTK (21% ± 7 SEM). Neither adenosine nor glutathione, present in UW, provided any increase in transduction when supplemented to HTK, although the addition of hydroxyethyl starch (HES) significantly improved transductions. To rule out size exclusion as a mechanism of HES, IGL-1 was tested but did not result in better transductions than HTK or DMEM. When supplemented to UW, anionic compounds reduced transduction, and this indicated a charge interaction mechanism of HES. In conclusion, this study demonstrates that effective vector delivery can be achieved under conditions of hypothermic liver perfusion. UW provides superior transduction to hepatocytes over nonstarch solutions.