Establishment of an intact functioning endothelial monolayer on a graft, at or near the time of implantation might be one of the ultimate requirement to get a biocompatible intravascular prosthesis. Following this hypothesis, endothelial cells from human stripped varicose veins were harvested with collagenase and grown on a human fibronectin matrix. The cultured cells (either in primary culture or throughout their lifespan in culture, 1 to 10 passages) exhibited characteristic cobblestone morphology and consistently displayed immunofluorescent staining for factor VUI-related antigen. Functional integrity of this endothelial cells was assayed by their ability to produce prostacyclin. After 15 minutes stimulation with 1 U/ml thrombin, production of 6 Keto PGF1α determined by Elisa was 17 ± 1.2 ng/106 cells. Proliferation was investigated in defined medium supplemented with various concentrations of serum (5 up Jto 30 %), ECGS (25 up to 150 μg/ml) and heparin (10−8 to 10−5M). Optimal growth required both, 100 μg/ml ECGS and 10−5M heparin, under these conditions cells culture achieved cell densities at confluence of 1.2 105 cells per square centimeter with doubling times of one day. Using I-heparin. a binding was demonstrated with an apparent Kd of 0.35 × 10−6 M. After characterization according to morphological, proliferative and functional criteria., cells were freezed at −80°C and ultimately used to coat polytetrafluoro-ethylene grafts (PTFE) which are currently used for vascular reconstructive surgery . Protein-treated material did allow cell attachment and growth to a confluent monolayer as assayed by light and scanning electron microscopy.These data suggested that i) stripped varicose veins provide a readily available source of adult human endothelial cell, ii) these cells grow vigorously in long-term culture when both ECGS and heparin are added to the culture medium, iii) they can adhere and grow to a confluent monolayer on vascular graft prior to implantation, iv) lining graft materials in vitro may be useful in improving the performance of small caliber vascular grafts according to prostacyclin production and surface bound heparin of these cells.
Secretion of prostacyclin, tissue plasminogen activator and its inhibitor by cultured adult human endothelial cells grown on different matrices