Dynamic Seeding of Perfusing Human Umbilical Vein Endothelial Cells (HUVECs) onto Dual-Function Cell Adhesion Ligands: Arg-Gly-Asp (RGD)−Streptavidin and Biotinylated Fibronectin

Abstract

Surfaces decorated with high affinity ligands can be used to facilitate rapid attachment of endothelial cells; however, standard equilibrium cell detachment studies are 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 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/cm2 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 α5β1 and αvβ3 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 α5β1 and αvβ3 integrins but that the combined ligands bound more total integrins than the two ligands tested separately.

Effects of electrode surface modification with chlorotoxin on patterning single glioma cells

“…The glioma cells patterned on the substrates with covalently-bound CTX demonstrated cell morphology extended beyond the gold electrodes (Fig 5b–c). Similar cell morphology was also observed when 9L glioma cells were patterned on substrates where gold electrodes were functionalized using the common cell adhesion ligands, such as chitosan, fibronectin, poly-L-lysine, and RGD 22, 23 (Fig. S1 in ESI).…”

“…5b and c). A similar cell morphology was also observed when 9L glioma cells were patterned on substrates where gold electrodes were functionalized using the common cell adhesion ligands, such as chitosan, fibronectin, poly-L-lysine, and RGD 22,23 (Fig. S1 in ESIw).…”

Capture of cervical exfoliative cells on a glass slide coated by 3-glycidyloxypropyl trimethoxysilane and poly-L-lysine

“…Glass slide is one of the most commonly used substrates for adhering cells which was completely transparent in a broad range of wavelengths employed. The modification of the glass substrates has been done in many reports by introduction of different terminal groups to adhere protein and cells [5] , [6] , [7] , [8] , [9] . Silane coupling regent is a kind of classical crosslinkers which has the ability to bind covalently to other molecules and even to biological molecules [10] , [11] which have amino, carboxyl or sulfhydryl groups [12] , [13] , [14] .…”

A new, rapid and reproducible method to obtain high quality endothelium in vitro

“…However, these cells in culture acquired variable morphologies and were not unequivocally characterized as HUVECs. Over-trypsinization is detrimental to cell viability as cells lose their capability to adhere to culture vessels (Anamelechi et al 2009). Furthermore, it can also result in detachment of other cells underlying the endothelium.…”