Seeding of Human Endothelial Cells on Valve Containing Aortic Mini-Roots: Development of a Seeding Device and Procedure

Abstract

Complete covering of an artificial valvular scaffold with endothelial cells may prevent thromboembolic complications and lead to an excellent biocompatibility. For this purpose, we developed a seeding device for reproducible cell seeding on valve containing aortic roots. Human endothelial cells and fibroblasts were obtained from saphenous vein pieces. Cryopreserved aortic roots (n = 25) were put into an especially developed tube, set on a rotator, and incubated with the cell suspension. The device rotated in two axes (sagittal and axial), ensuring slight movements of the leaflets. The rotation alternated with resting periods, allowing cell attachment to the surface. Different resting periods were tested (groups 1, 2, and 3 were 30, 45, and 60 min, respectively; n = 5 each). Total incubation time was 24 hours followed by further culturing for 6 days. In two further groups (groups 4 and 5; n = 5 each), a modified inlay was used to allow the cell suspension to flow around the entire graft. In group 4 the grafts were again incubated with human endothelial cells; however, in group 5 pre-seeding with autologous fibroblasts was done in addition. Immunohistochemical staining with antibodies against factor VIII, CD31, laminin, collagen IV, and CD90 were done, and scanning electron microscopy was done after initial seeding and after 6 days in culture. Seeding resulted in homogenous cell layers on the luminal surface of the free walls in all groups. With resting periods of 45 minutes, these results were also obtained on the leaflets, whereas the other resting times resulted in defects of the endothelial cell layer on the cusps. After 6 days under culture conditions, the endothelial cell layers were confluent and viable, with the exception of the leaflets in group 1. With the modified inlay (groups 4 and 5), confluent cell layers were also achieved on the outer surface. In group 5 pre-seeding with autologous fibroblasts resulted in enhanced synthesis of extracellular matrix proteins, as was demonstrated with immunohistochemical staining for collagen IV and laminin. With this newly developed seeding device, confluent cell layers on valve containing aortic roots were reproducibly achieved. The technique enables further experimental research and even clinical application.