Reduced glutathione levels and expression of the enzymes of glutathione synthesis in cryopreserved hepatocyte monolayer cultures

Cryopreservation of monolayers of hepatocytes in a freezing medium containing 10% (v/v) dimethylsulfoxide, 90% (v/v) foetal calf serum retains cell morphology and viability, but cells lose up to 50% of their intracellular reduced glutathione. This is accompanied by a small increase in glutamate cysteine ligase expression in cryopreserved cultures, but glutathione synthetase expression is undetectable post-cryopreservation. Inclusion of ascorbic acid and alpha-tocopherol in the freezing medium improves maintenance of reduced glutathione content post-cryopreservation at 84% of the levels in non-cryopre served monolayer cultures, but does not restore glutathione synthetase expression. The inability to synthesise reduced glutathione will mean that cryopreserved hepatocyte monolayers are more susceptible to toxic insults. (c) 2006 Elsevier Ltd. All rights reserved.