New approach to improving endothelial preservation in cryopreserved arterial substitutes

Abstract

The endothelial loss provoked by the methods of vascular cryopreservation used at most human vessel banks is one of the main factors leading to the failure of grafting procedures performed using cryopreserved vessel substitutes. This study evaluates the effects of the storage temperature and thawing protocol on the endothelial cell loss suffered by cryopreserved vessels, and optimises the thawing temperature and protocol for cryopreserving arterial grafts in terms of that producing least endothelial loss. Segments of the common iliac artery of the minipig ( n=20) were frozen at a temperature reduction rate of 1 °C/min in a biological freezer. After storing the arterial fragments for 30 days, study groups were established according to the storage temperature (−80, −145 or −196 °C) and subsequent thawing procedure (slow or rapid thawing). Fresh vessel segments served as the control group. Once thawed, the specimens were examined by light, transmission, and scanning electron microscopy. The covered endothelial surface was determined by image analysis. Data for the different groups were compared by one way ANOVA. When cryopreservation at each of the storage temperatures was followed by slow thawing, the endothelial cells showed improved morphological features and viability over those of specimens subjected to rapid thawing. Rapidly thawed endothelial cells showed irreversible ultrastructural damage such as mitochondrial dilation and rupture, reticular fragmentation, and peripheral nuclear condensation. In contrast, slow thawing gave rise to changes compatible with reversible damage in a large proportion of the endothelial cells: general swelling, reticular dilation, mitochondrial swelling, and nuclear chromatin condensation. Gradually thawed cryopreserved arteries showed a lower proportion of damaged cells identified by the TUNEL method compared to the corresponding rapidly thawed specimens ( p<0.05, for all temperatures). In all the groups in which vessels underwent rapid thawing (except at −145 °C), significant differences ( p<0.05) in endothelial cover values were recorded with respect to control groups. Storage of cryopreserved vessels at −80 °C followed by rapid thawing led to greatest endothelial cell loss (61.36 ± 9.06% covered endothelial surface), while a temperature of −145 °C followed by slow thawing was best at preserving the endothelium of the vessel wall (89.38 ± 16.67% surface cover). In conclusion, storage at a temperature of −145 °C in nitrogen vapour followed by gradual automated thawing seems to be the best way of preserving the endothelial surface of the arterial cryograft. This method gives rise to best endothelial cell viability and cover values, with obvious benefits for subsequent grafting.