ID: mamprin Abstract No.: 158 Volume Regulation and mRNA Expression of Aaquaporine 8 in Hepatocytes Cold Stored at 0◦C in Preservation Solutions Maŕia Mamprin, Cristina Bellarosa, Silvana Petrocelli, Edgardo Guibert, Claudio Tiribelli, Joaquin Rodŕiguez a Ciencias Fisiologicas, Facultad de Ciencias Bioquimicas y Farmaceuticas. Universidad Nacional de Rosario. UNESCO Chair in Cryobiology, S2002LRK Rosario, Argentina b Ciencias Biologicas, Facultad de Ciencias Bioquimicas y Farmaceuticas. Universidad Nacional de Rosario. UNESCO Chair in Cryobiology, S2002LRK Rosario, Argentina d BBCM, Centro Studi Fegato, AREA Science Park, Basovizza SS 14 Km 136.5. University of Trieste, 34012 Trieste, Italy Different storage solutions, such as the University of Wisconsin solutions (UW) have been successfully used for the temporal maintenance of the liver of animals and humans. Many studies have demonstrated the effectiveness of cold storage of hepatocytes and successful application on Bioartificial liver devices and hepatocellular transplantation. The aim of the present study was to investigate the effect of cold storage time at 0◦C with two different preservation solutions: UW and a novel BES-based solution (BGS), on gene expression (at mRNA level for Aquaporine 8 (AQP8)) and water movements of isolated rat hepatocytes. Hepatocytes were isolated from male Wistar rats by collagenase perfusion and cold stored for 72 h at 0◦C in either UW or a 70 mM BES solution containing 50mM sucrose and 100mM potassium gluconate. Total RNA was isolated from hepatocytes taken at different time intervals. Real time RT-PCR was used to quantify the AQP8 mRNA in freshly isolated hepatocytes and after 72 h of cold storage and during a subsequent 2 h rewarming period; s-actin was used as housekeeping gene to normalize the expression of target genes. For the AQP8 (accession bank: NM 019158) we used Upper primer: 5-TTG GGG CTC ATC ATT GCT AC-3, position 333; and Lower primer: 5-AAG GCC TCC AAC CAG TGT GA-3, position 403. To evaluate the water content of cells during preservation and rewarming, the [wet weight dry weight] / dry weight of hepatocytes collected at different time intervals was determinate after 48 h desiccation at 90◦C. To monitor the evolution of hepatocyte water content (Vend / Vinitial), each volume (Vend) was normalized to the volume of hepatocyte at the corresponding time of control cells at 37◦C (Vinitial). Cold storage induced a significant hepatocyte shrinkage at 72 h: UW 0.56 ± 0.03; BCG: 0.87 ± 0.13 vs. Control 1.00 (n=10). On the contrary, no changes in expression of AQP8 at mRNA level was found During the rewarming, cells preserved in both solutions recover a volume similar to the control group (control: 1.15 ± 0.06; UW: 1.03 ± 0.09, BCG: 1.21 ± 0.12, n=10). From these data we conclude that BES-based solution is as effective as UW solution for hepatocyte preservation. The change in cell volume observed both during cold storage and after rewarming is not related to change in the mRNA levels of AQP8. Irrespective of the cell shrinkage observed at 0◦C, hepatocytes recover a normal cellular volume during the rewarming pointing to the possibility to safety preserve the cells in both solutions for further use. Conflict of interest: None declared Source of funding: Ministero degli Affari Esteri, Italy. Progetti di Grande Rilevanza Prot. 269/P/0114337. ANPCyT, Argentina, PICT 03-14492. UNESCO Chair in Cryobiology. Cryo 2006 www.cryo2006.org sputtek@uke.uni-hamburg.de