67 GLUTATHIONE ADDED TO CULTURE MEDIUM AND CRYOPROTECTANT SOLUTION ENHANCE THE VIABILITY OF EMBRYONIC STEM CELLS AFTER THAWING
G. A. Kim A , S. T. Lee A , E. J. Lee A , J. H. Choi A and J. M. Lim AGamete and Stem Cell Biotechnology Laboratory, Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea
Reproduction, Fertility and Development 21(1) 133-134 https://doi.org/10.1071/RDv21n1Ab67
Published: 9 December 2008
Abstract
We have attempted to improve cell viability of embryonic stem cells (ESC) after freezing and thawing, and, as a result, an efficient cryoprotectant for cryopreserving of mouse ESC was selected in a previous study. This study was subsequently conducted to examine whether the addition of glutathione (GSH), an antioxidant, to the optimized culture medium and cryoprotectant solution could improve post-thaw cell viability of ESC. Embryonic stem cells of a mouse E14 cell line were subcultured 5 times and subsequently employed for the cryopreservation. Dulbecco’s minimal essential medium, to which 100 μm GSH was added or not, was used as a basal medium for ESC culture and a freezing solution. Dimethyl sulfoxide (10%, v/v) and ethylene glycol (10%, v/v) were used as cryoprotectants. In experiment 1, intracytoplasmic concentrations of reactive oxygen species (ROS) in ESCs subcultured and cryopreserved in GSH-free and GSH-containing medium was measured immediately after cryopreservation. A significant (P < 0.0001) decrease in ROS level was detected in ESC cultured and cryopreserved in GSH-containing medium compared with GSH-free medium. Cell viability was subsequently assessed immediately and 72 h after thawing in experiment 2. Improved post-thaw viability was detected when GSH-containing media were employed. There were no significant differences of the viability between fresh and frozen–thawed ESC, regardless of time of assessment immediately (P > 0.1325; 0.653 v. 0.604) and 72 h (P > 0.2998; 0.671 v. 0.626) after treatment. However, there was lower post-thaw viability at 0 h (P < 0.0024; 0.671 v. 0.544) and at 72 h (P < 0.0138; 0.742 v. 0.59) in ESC cultured and cryopreserved in GSH-free medium than that in ESC cultured and cryopreserved with GSH-containing medium. These results demonstrated that use of GSH as an antioxidant affects the level of ROS and promotes post-thaw survival of frozen ESC.
