Quantification of vascular graft seeding by use of computer-assisted image analysis and genetically modified endothelial cells

Abstract

Current methods for the evaluation of retention of endothelial cells seeded on vascular grafts are limited by the inability to specifically identify and quantitate seeded cells on a long-term basis. To address this problem we developed a method of quantification of graft surface coverage using genetic labeling of endothelial cells combined with computer-assisted image analysis. Rabbit aortic endothelial cells were transduced with a marker gene (lac-Z) and seeded on polytetrafluoroethylene grafts. After histochemical staining in which the genetically labeled cells turn blue, computer-assisted image analysis was used to measure the percentage of graft surface covered by the seeded cells. The utility of the method was evaluated by using it to assess the effect on graft coverage of seeded cell density and by precoating with fibronectin. Quantification of surface area coverage was automated and reproducible both between scans and between observers. Use of this method allowed the determination of a linear correlation between cell density in the seeding suspension and graft coverage (r2 = 0.93, p less than 0.0001). The method also permitted confirmation of the positive contribution of fibronectin coating to graft coverage by seeded cells: 73% coverage coated versus 8% coverage uncoated (p less than 0.0001). The ability of this method to specifically identify genetically marked endothelial cells and their progeny makes it attractive for use in studies targeted at optimization of graft coverage in vivo.

Vascular Grafts and the Endothelium

“…The total number of extractable endothelial cells compares very favorably to that from human external jugular vein, forearm veins, or the saphenous vein (when unsuitable for grafting) where the maximum number of endothelial cells obtained has been 0.56-100 × 10 5 cells [106]. Having an abundant supply of endothelial cells is crucial because cell concentration in the seeding suspension correlates with graft coverage [113]. For example, although 73% graft coverage could be achieved with 2 × 10 5 cells/cm 2 , coverage was only 35% when 7 × 10 4 cells/cm 2 were used [113].…”

“…Having an abundant supply of endothelial cells is crucial because cell concentration in the seeding suspension correlates with graft coverage [113]. For example, although 73% graft coverage could be achieved with 2 × 10 5 cells/cm 2 , coverage was only 35% when 7 × 10 4 cells/cm 2 were used [113]. The current optimum values that maximize attachment and retention are 4-16 × 10 5 cells/cm 2 for the various synthetic grafts [114,115].…”

Cellular engineering of vascular bypass grafts: Role of chemical coatings for enhancing endothelial cell attachment

“…Overall, fibronectin has also been shown to have much better cellular attachment and retention than uncoated grafts ofePTFE, dacron and MyoLink (GIUDICEANDREA et al, 1998;KENT et al, 1992;KESLER et al, 1986;NEWMAN et al, 1991;SCOTT and MANN, 1990;WALLUSCHECK et al, 1996a). in comparative studies, FN shows enhanced cell attachment in comparison with collagen (BUDDet al, 1989;THOMSON et al, 1989), gelatin (BUDDet aL, 1989;IVARSSON et al, 1989;THOMSON et aL, 1989), poly-l-lysin (BUDD et al, 1989), laminin, albumin and gelatin (HASSON et aL, 1986;PRONK et al, 1994;THOMSON et al, 1989).…”

“…Low seeding densities are not optimum and again show poor cell retention on exposure to physiological flow (HAEGERSTRAND et al, 1993;MIYATA et al, 1991;NEWMAN et al, 1991). This leads to poor clinical patency (HERRING et al, 1994;ZILLA et al, 1989).…”