Seeding of Endothelial Cells on the Luminal Surface of a Sheet Model of Cold-Stored (at 4°C) Sheep Carotid Arteries

Abstract

Cold-stored arteries are biomaterials that potentially represent an abundant "off-the-shelf" source of vascular grafts for use in vascular surgery. One of the keys to reestablishing the antithrombogenic endothelial cell (EC) lining of cold-stored arterial grafts is to maximize the number of ECs that attach following seeding. In this study, the cold-stored sheep carotid artery is used as a substrate to determine the conditions that maximize EC adherence following seeding. The effect of serum concentration, duration of seeding incubation, seeding density, and period of cold storage on attachment of ECs following seeding of 4-week cold-stored sheep carotid arteries (n = 5 arteries), 8-week cold-stored sheep carotid arteries (n = 5 arteries), and 12-week cold-stored sheep carotid arteries (n = 5 arteries) was examined. Three experiments (serum concentration, time of incubation, and seeding density) were conducted to determine the conditions that maximized the number of cultured sheep carotid artery ECs that attached to cold-stored sheep carotid artery following seeding. A flat sheet model was used. Serum concentration (0%, 10%, 20%, and 30%) in the seeding suspension did not have a significant effect on overall EC adherence on 4-, 8-, and 12-week cold-stored arteries. Time of seeding incubation (30, 60, and 90 min) did not have a significant effect on overall EC adherence on 4-, 8-, and 12-week cold-stored arteries. Seeding density (500,000 cells/ml, 1,000,000 cells/ml, and 2,000,000 cells/ml) had a significant effect (p = 0.036) on overall EC adherence on 4-, 8-, and 12-week cold-stored arteries. The period of cold storage (4, 8, and 12 weeks) of the artery had a significant effect (p = 0.002, p < 0.0001, p < 0.0001 in serum, time, and seeding density experiments, respectively) on overall EC adherence following seeding. Pairwise comparisons of EC adherence revealed the following. In the serum experiment, EC adherence on 4-week cold-stored arteries was significantly greater than on 8-week cold-stored arteries (p = 0.003) and 12-week cold-stored arteries (p = 0.002). This effect was due largely to the significant difference between EC adherence on 4-week and 8-week cold-stored arteries (p = 0.0002) and between EC adherence on 4-week and 12-week cold-stored arteries (p = 0.0091) at 20% serum. In the time experiment, EC adherence on 4-week cold-stored arteries was significantly greater than on 12-week cold-stored arteries (p < 0.0001). In the seeding density experiment, EC adherence on 4-week cold-stored arteries was significantly greater than on 8-week cold-stored arteries (p < 0.0001) and 12-week cold-stored arteries (p < 0.0001). In the same experiment, EC adherence following seeding at a density of 1,000,000 cells/ml and 2,000,000 cells/ml was significantly greater (p = 0.03 and p = 0.02, respectively) than EC adherence following seeding at a density of 500,000 cells/ml. Thus, it was determined that 4-week cold-stored arteries were superior to 8- and 12-week cold-stored arteries in terms of the number of ECs that adhered. It was also determined that a seeding density of 1,000,000 or 2,000,000 cells/ml was superior to a seeding density of 500,000 cells/ml in terms of producing maximal EC attachment. The ideal conditions, from those examined, for maximizing EC attachment to cold-stored arteries were 4 weeks of cold storage, a serum concentration of 20%, a seeding density of 2,000,000 cells/ml, and a duration of incubation of 30-90 min.