In vitro lining of fibronectin coated PTFE grafts with cryopreserved saphenous vein endothelial cells.

In an attempt to produce instant endothelial cell (EC) monolayers on graft surfaces, cryopreserved cultivated human saphenous vein endothelial cells (HSVEC) were cultivated on reinforced PTFE prostheses. The graft surface was precoated with 40 micrograms/ml human fibronectin (HFN) prior to seeding with 200 x 10(3) EC/cm2. The seeding procedure was performed in a specially designed rotation device. After a cultivation period of 9 days, the seeded endothelial cells on the PTFE prostheses were exposed to shear stresses in a perfusion circuit, containing a bubble oxygenator, a roller pump and a tygon perfusion loop. The applied shear forces were 3 and 6 dyn/cm2, respectively. In control grafts, spontaneous cell detachment occurred from day 11 onwards and only 50% of the graft surface remained endothelialized on day 16. When the grafts were exposed to 3 dyn/cm2 only small cell-free areas less than 1000 micron in diameter were found after 4 hours of perfusion. In contrast, exposure to 6 dyn/cm2 produced discouraging results: as early as 4 hours after the onset of perfusion 50% of the graft surface was cell free. After 16 hours only 20% endothelial cell coverage was seen under the stereo microscope. However, assuming that an ideal precoating substratum can be found, the two stage technique of in vitro endothelialization of vascular grafts with autologous endothelial cells may offer a promising clinical method. Moreover, the fact that our grafts were lined with cryopreserved EC implies the possible prospect of cell banks supplying unlimited numbers of EC for subsequent bypass operations.