Flow cytometric analysis of endothelial cell viability in arterial allograft

Abstract

Arterial allografts have known advantages over prosthetic vascular conduit for treatment of heart valvular disease, congenital heart disease, and aortic disease. Cell viability may play a role in determining the long-term outcome of allografts. Endothelial cell is one important part in determining the allograft viability. To evaluate the viability of endothelial cells using current allograft preservation techniques, porcine heart valve leaflets and arterial wall were subjected to collagenase digestion. Single endothelial cell suspension was labeled with Griffonia simplicifolia agglutinin-fluorescein isothiocyanate (GSA-FITC), a vascular endothelial cell specific marker. The cell suspension was washed and incubated with propidium iodide (PI), which does not bind with viable cells. Endothelial cell viability was evaluated by calculating the percentage of the GSA-FITC(+) and PI(−) groups using flowcytometric analysis. For sterilization, allografts were treated with 4°C antibiotic solution for 24 hours. After this, half of the allografts were stored in 4°C RPMI 1640 with HEPES buffer culture medium with 10% fetal bovine serum for 1–14 days (Group I). The other half of the allografts was cryopreserved with a currently used technique (Group II). During the procurement and sterilization, 22.8% and 24.4% of endothelial cell viability declined, respectively. In Group I, 11.9% of endothelial cell viability steadily declined further during 14 days of storage. In Group II, 13.7% of endothelial cell viability declined. These results show that the largest loss of endothelial cell viability occurs during the initial process. After 14 days of storage under 4°C nutrient medium or cryopreservation, about 40% of endothelial cell viability is maintained. There were no differences among the endothelial cell viability from aortic valve leaflet, pulmonic valve leaflets, aortic wall, and pulmonic wall.