Surfaces decorated with high affinity ligands can be used to facilitate rapid attachment of endothelial cells: however. standard equilibrium cell detachment Studies Lire poorly suited for assessing these initial adhesion events. Here, a dynamic seeding and cell retention method was used to examine the initial attachment of perfusing human umbilical vein endothelial cells (HUVECs) to bare Teflon-AF substrates, Substrates pre-adsorbed with fibronectin alone, or Substrates co-pre-adsorbed with two dual-function it cell-adhesion ligands: biotinylated fibronectin (bFN) and RGD-streptavidin mutant (RGD-SA). Cell attachment was evaluated as a function of cell trypsinization (integrin digestion), surface protein formulation, and cell perfusion rate. Surfaces co-pre-adsorbed with bFN and RGD-SA showed the highest density of attached cells after 8 min of perfusion and the highest percent retention When Subjected to shear flow at 60 dynes/cm(2) for 2 min. Surfaces with no ligand treatment showed the lowest cell attachment and retention under flow in all cases. HUVECs trypsinized with mild 0.025% trypsin/ethylenediaminetetraacetic acid (EDTA) showed greater cell adhesion after perfusion and higher percent retention after shear flow than those trypsinized Using harsher 0.05% trypsin/EDTA. The preferential affinities of the two dual-function ligands for alpha(5)beta(1) and alpha(v)beta(3) integrins were also examined by surface plasmon resonance (SPR) spectroscopy. The dynamic cell seeding Studies confirmed that the dual-function ligand system promotes HUVEC adhesion and retention at short time points when tested using a perfusion assay. SPR studies showed that the two ligands exhibited equal affinity for both alpha(5)beta(1) and alpha(v)beta(3) integrins but that the combined ligands bound more total integrins than the two ligands tested separately.