Surface endothelialization could improve the long-term performance of vascular grafts and stents. We previously demonstrated that aerosol-generated fibronectin-derived peptide micropatterns consisting of GRGDS spots over a WQPPRARI background increased endothelial cell yields in static cultures. We developed a novel fluorophore-tagged RGD peptide (RGD-TAMRA) to visualize cellsurface interactions in real-time. Here, we studied the dynamics of endothelial cell response to laminar flow on these peptide-functionalized surfaces. Endothelial cells were exposed to 22 dyn/cm 2 wall shear stress while acquiring time-lapse images. Cell surface coverage and cell alignment were quantified by undecimated wavelet transform multivariate image analysis. Similar to gelatin-coated surfaces, surfaces with uniform RGD-TAMRA distribution led to cell retention and rapid cell alignment (˜63% of the final cell alignment reached within 1.5 hours), contrary to the micropatterned surfaces. The RGD-TAMRA peptide is a promising candidate for endothelial cell retention under flow while the spray-based micropatterned surfaces are more promising for static cultures.

The expansion of human peripheral blood endothelial progenitor cells to obtain therapeutically relevant endothelial colony-forming cells (ECFCs) has been commonly performed on xeno-derived extracellular matrix proteins. For cellular therapy applications, xeno-free culture conditions are desirable to improve product safety and reduce process variability. We have previously described a novel fluorophore-tagged RGD peptide (RGD-TAMRA) that enhanced the adhesion of mature endothelial cells in vitro. To investigate whether this peptide can replace animal-derived extracellular matrix proteins in the isolation and expansion of ECFCs, peripheral blood mononuclear cells from 22 healthy adult donors were seeded on RGD-TAMRA-modified polystyrene culture surfaces.Endothelial colony formation was significantly enhanced on RGD-TAMRA-modified surfaces compared to the unmodified control. No phenotypic differences were detected between ECFCs obtained on RGD-TAMRA compared to ECFCs obtained on rat-tail collagen-coated surfaces. Compared with collagen-coated surfaces and unmodified surfaces, RGD-TAMRA surfaces promoted ECFC adhesion, cell spreading, and clonal expansion. This study presents a platform that allows for a comprehensive in vitro evaluation of peptide-based biofunctionalization as a promising avenue for ex vivo ECFC expansion. K E Y W O R D Sendothelial progenitor cells, ex vivo expansion, extracellular matrix-derived peptides, surface modification, vascular regeneration, xeno-free

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