Rat hepatocyte culture model of macrosteatosis: effect of macrosteatosis induction and reversal on viability and liver-specific function.

BACKGROUND & AIMS: A common cause of liver donor ineligibility is macrosteatosis. Recovery of such livers could enhance donor availability. Living donor studies have shown diet-induced reduction of macrosteatosis enables transplantation. However, cadaveric liver macrosteatotic reduction must be performed ex vivo within hours. Towards this goal, we investigated the effect of accelerated macrosteatosis reduction on hepatocyte viability and function using a novel system of macrosteatotic hepatocytes. METHODS: Hepatocytes isolated from lean Zucker rats were cultured in a collagen sandwich, incubated for 6 days in fatty acid-supplemented medium to induce steatosis, and then switched for 2 days to medium supplemented with lipid metabolism promoting agents. Intracellular lipid droplet size distribution and triglyceride, viability, albumin and urea secretion, and bile canalicular function were measured. RESULTS: Fatty acid-supplemented medium induced microsteatosis in 3 days and macrosteatosis in 6 days, the latter evidenced by large lipid droplets dislocating the nucleus to the cell periphery. Macrosteatosis significantly impaired all functions tested. Macrosteatosis decreased upon returning hepatocytes to standard medium, and the rate of decrease was 4-fold faster with supplemented agents, yielding 80% reduction in 2 days. Viability of macrosteatosis reduced hepatocytes was similar to control lean cells. Accelerated macrosteatotic reduction led to faster recovery of urea secretion and bile canalicular function, but not of albumin secretion. CONCLUSIONS: Macrosteatosis reversibly decreases hepatocyte function and supplementary agents accelerate macrosteatosis reduction and some functional restoration with no effect on viability. This in vitro model may be useful to screen agents for macrosteatotic reduction in livers before transplantation.

Hydroxyethyl starch-based preservation solutions enhance gene therapy vector delivery under hypothermic conditions.

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 degrees C or 37 degrees 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 degrees 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.