Cryopreservation of Human Hepatocytes Alters the Mitochondrial Respiratory Chain Complex 1

Abstract

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.

Improved cryopreservation of human hepatocytes using a new xeno free cryoprotectant solution

“…However, in our small pilot study where we compared both freezers, we did not find using the CRF better than the ordinary -70 ℃ freezer. These findings were supported in reports by others [20,22,23] where no difference was shown between CRF, the Nalgene propan-2-ol device or simply using -20 ℃ and -70 ℃ freezers. The aim of our study was to compare the efficacy of two complete cryopreservation protocols, CB protocol and DMSO-UW protocol.…”

Serum- and albumin-free cryopreservation of endothelial monolayers with a new solution

“…14,35,36 Loss of mitochondrial membrane potential was also described after cryopreservation, for example in sperm, 37–39 mouse embryos, 40 muscle, 41,42 hepatocytes 43 and in isolated mitochondria. 44,45 Also, ultrastructural changes in mitochondria, like mitochondrial swelling and rupture have been described to occur in endothelial cells after vascular cryopreservation, 46,47 however, little is known about mitochondrial membrane potential in this cell type.…”

Adult Stem Cell Therapy in Chronic Liver Diseases

“…However, human hepatocytes have been shown to be unsuitable as the availability of human livers as a source of cells is limited, and it is difficult to maintain hepatocyte viability and function during cryopreservation of the liver. In addition, hepatocytes are difficult to culture in vitro [6]. Moreover, while the safe transplantation of -5% of the total recipient liver cell mass (a maximum of 1-2×10 8 hepatocytes/body weight…”