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-cryopreserved 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.
Stevenson
David J
DJ
Bioengineering Unit, University of Strathclyde, Wolfson Centre, 106 Rottenrow, Glasgow G4 0NW, UK.
Morgan
Caroline
C
McLellan
Lesley I
LI
Helen Grant
M
M
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D005721:Glutamate-Cysteine Ligase / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D005981:Glutathione Synthase / Q000378:metabolism*; D022781:Hepatocytes; D008297:Male; D018613:Microscopy, Confocal; D051381:Rats; D017207:Rats, Sprague-Dawley; D024502:alpha-Tocopherol / Q000494:pharmacology
D003451:Cryoprotective Agents; D005721:Glutamate-Cysteine Ligase; D005981:Glutathione Synthase; D005978:Glutathione; D024502:alpha-Tocopherol; D001205:Ascorbic Acid