Long-Term Cryopreservation of Autologous Veins in Rabbits

Abstract

This study was undertaken to determine the effect of long-term cryopreservation on graft ultrastructure and endothelial cell viability in an animal model. The jugular veins from 12 New Zealand White rabbits were excised with a ‘no-touch’ technique and divided into four groups: control group (fresh veins); group 1, veins cryopreserved for 1 month; group 2, veins cryopreserved for 2 months; and group 3, veins cryopreserved for 3 months. Cryopreservation was accomplished by rapid freezing (−5°Cs⁻¹ to −196°C) in a solution of 17.5% dimethylsulphoxide and 20% fetal bovine serum and by storage in liquid nitrogen. Veins were then implanted as a carotid autograft (three grafts/group). At the time of graft implantation a segment of the paired matched vein was perfusion-fixed and evaluated by scanning and transmission electron microscopy, whereas the remainder were subjected to endothelial cell culture techniques to determine cell viability. Autografts were removed 1 month after implantation and subjected to similar evaluations. Histological changes seen in cryopreserved veins were dependent on preservation time and included focal endothelial cell blebbing, cytoplasmic vacuolization and disruption of cell-to-cell contacts. Smooth muscle cells showed mithocondrial swelling. Patency was identical in all groups (66.6%). Explants at 1 month were similar in histological appearance to fresh veins with a smooth endothelial cell lining arranged longitudinally and intact cell junctions. Endothelial cells could be cultured from fresh veins and 1-month-old explants but not from the cryopreserved graft surface before implantation. The present technique of cryopreservation leads to some damage of graft architecture and loss of endothelial cell viability. However, these changes are reversed after 1 month of implantation at which time a viable cell lining is restored.