67 GLUTATHIONE ADDED TO CULTURE MEDIUM AND CRYOPROTECTANT SOLUTION ENHANCE THE VIABILITY OF EMBRYONIC STEM CELLS AFTER THAWING

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 0.1325; 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.