Cryopreservation of human hepatocytes alters the mitochondrial respiratory chain complex 1.
Transplantation of human hepatocytes has recently been demonstrated as a safe alternative to partially correct liver inborn errors of metabolism. Cryopreservation remains the most appropriate way of cell banking. However, mitochondrial-mediated apoptosis has been reported after cryopreservation and little is known on the involved molecular mechanisms. The aim of this study was to investigate mitochondrial functions of freshly isolated and cryopreserved/thawed hepatocytes from mice and humans. We report here that cryopreservation induced a dramatic drop of ATP levels in hepatocytes. The oxygen consumption rate of cryopreserved/ thawed hepatocytes was significantly lower compared to fresh cells. In addition, the uncoupling effect of 2,4-dinitrophenol was lost, in parallel with a reduction of mitochondrial membrane potential. Furthermore, a decrease in mitochondrial respiratory rate was evidenced on permeabilized hepatocytes in the presence of substrate for the respiratory chain complex 1. Interestingly, this effect was less marked with a substrate for complex 2. Electron microscopy examination indicated that mitochondria were swollen and devoid of cristae after cryopreservation. These changes could explain the cytosolic release of the proapoptotic protein cytochrome c in cryopreserved cells. Nevertheless, no caspase 9-3 activation and only few apoptotic and necrotic cells were found, indicating that the subsequent cell death program was not yet evidenced. Our results demonstrate that cryopreservation of hepatocytes induced alteration of the mitochondrial machinery. They also suggest that, in addition to technical progress in the cryopreservation procedure, protection of the respiratory chain complex 1 should be considered to improve the quality of cryopreserved hepatocytes.
Laboratory of Paediatric Hepatology and Cell Therapy, Université Catholique de Louvain, Cliniques St Luc, Brussels, Belgium.
Xavier
X
Stéphenne
Mustapha
M
Najimi
Dung Khuu
DK
Ngoc
Françoise
F
Smets
Louis
L
Hue
Bruno
B
Guigas
Etienne M
EM
Sokal
D019297:2,4-Dinitrophenol / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival; D015925:Cryopreservation; D045304:Cytochromes c / Q000378:metabolism; D042967:Electron Transport Complex I / Q000378:metabolism*; D004789:Enzyme Activation; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D008810:Mice, Inbred C57BL; D046529:Microscopy, Electron, Transmission; D008930:Mitochondria, Liver / Q000378:metabolism / Q000648:ultrastructure; D010101:Oxygen Consumption; D014475:Uncoupling Agents / Q000494:pharmacology
D014475:Uncoupling Agents; D000255:Adenosine Triphosphate; D045304:Cytochromes c; D020169:Caspases; D042967:Electron Transport Complex I; D019297:2,4-Dinitrophenol