Freezing and Thawing Conditions of Rat Hepatocytes.
- Author:
Seong Gyu HWANG
1
;
Eun Mi JUNG
;
Seong Wook OH
;
Chang Il KWON
;
Kyung Chul KIM
;
Sung Pyo HONG
;
Pil Won PARK
;
Kyu Sung RIM
Author Information
1. Department of Internal Medicine, Pochon CHA University College of Medicine, Sungnam, Korea. sghwang@cha.ac.kr
- Publication Type:Original Article
- Keywords:
Cryopreservation;
Hepatocyte;
Freezing-thawing;
Cell culture
- MeSH:
Animals;
Cell Count;
Cell Culture Techniques;
Cell Survival;
Centrifugation, Density Gradient;
Collagen Type I;
Cryopreservation;
Dimethyl Sulfoxide;
Freezing*;
Glycerol;
Hepatocytes*;
Iohexol;
Liver, Artificial;
Nitrogen;
Rats*;
Shock;
Trypan Blue
- From:The Korean Journal of Hepatology
2001;7(3):308-314
- CountryRepublic of Korea
- Language:Korean
-
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.
