Freezing and Thawing Conditions of Rat Hepatocytes

Hwang, Seong Gyu; Jung, Eun Mi; Oh, Seong Wook; Kwon, Chang Il; Kim, Kyung Chul; Hong, Sung Pyo; Park, Pil Won; Rim, Kyu Sung

Abstract

BACKGROUND/AIMS: During cryopreservation of hepatocytes, a dramatic loss in cell number, viability and differentiated cell function is usually inevitable because hepatocytes are very sensitive to stress during freezing and thawing. We tried to investigate the optimal cryopreservation conditions of hepatocytes including the constituents of the freezing medium and freezing rate.
METHODS
Isolated hepatocytes were cryopreserved in media containing 10% glycerol or dimethyl sulfoxide (DMSO) of variable concentration. Different freezing procedures (stepwise, rapid, and programmed with or without shock cooling) were used and they were stored in a liquid nitrogen tank. After rapid thawing at 39degrees C, followed by dilution and removal of the cryopreservative, the ability of the hepatocytes to exclude trypan blue dye (TB) was evaluated. Hepatocytes were fractionated through a Nycodenz density gradient centrifugation (DGC) to eliminate dead cells. Cells were plated on dishes coated with type I collagen.
RESULTS
Cell viability of hepatocytes recovered from cryopreservation was maintained better using 10, 15, and 20% DMSO as a cryopreservative and programmed cell freezer with shock cooling. After Nycodenz DGC a hepatocyte fraction highly enriched in viable cells could be taken between 11% and 30%. In culture, cryopreserved hepatocytes exhibited a morphology with epithelial characteristics.
CONCLUSIONS
These results suggest that rate-adjusted programmed freezing with shock cooling and 10, 15 and 20% DMSO increased the viability of cryopreserved hepatocytes. The hepatocyte fraction highly enriched in viable cells could be taken using Nycodenz DGC. In order to establish a bank of hepatocytes for hepatocyte transplantations and artificial livers a more improved method is nevertheless necessary to increase the viability of hepatocytes after cryopreservation.