Evaluating the Effects of Dithiothreitol and Fructose on Cell Viability and Function of Cryopreserved Primary Rat Hepatocytes and HepG2 Cell Line

BACKGROUND: Hepatocytes are used as an in vitro model to evaluate drug metabolism. Human hepatocyte transplant has been considered as the temporary treatment of acute liver failure. Optimization freezing methods is very important to preserve both cell viability and function which are achieved by cryopreservation mostly always. OBJECTIVES: The present study aimed to investigate the cryoprotective effect of DTT and fructose on primary rat hepatocytes and HepG2 cells. MATERIALS AND METHODS: Both fresh rat hepatocytes and HepG2 cell line were incubated with fructose (100 and 200 mM) and dithiothreitol (DTT) (25, 50, 100, 250, and 500 μM) at 37°C for 1 and 3 hours, respectively. The preincubated hepatocytes were cryopreserved for two weeks. Hepatocytes viability and function were determined post thawing and the results were compared with the control group. RESULTS: The viability of both rat hepatocytes and HepG2 cells were significantly increased after one hour preincubation with fructose 200 mM. Preincubation with DTT (50 μM, 100 μM. 250 μM and 500 μM) improved the viability and function upon thawing in both cell types (P < 0.001). In rat hepatocytes, no significant change was observed in albumin, urea production, and LDH leakage after preincubation with fructose or DTT. In HepG2 cells, albumin and urea production were significantly increased after preincubation with DTT (500 μM, 1 hour). The GSH content was significantly increased in DTT (250 and 500 μM, 1 hour) groups in both rat hepatocyte and HepG2 cells. CONCLUSIONS: Incubation of hepatocytes with fructose and DTT prior to the cryopreservation can increase the cell viability and function after thawing.