Effect of cell-seeding density on the proliferation and gene expression profile of human umbilical vein endothelial cells within ex vivo culture.

BACKGROUND AIMS: Characterization of endothelial cell-biomaterial interaction is crucial for the development of blood-contacting biomedical devices and implants. However, a crucial parameter that has largely been overlooked is the cell-seeding density. METHODS: This study investigated how varying cell-seeding density influences human umbilical vein endothelial cell (HUVEC) proliferation on three different substrata: gelatin, tissue culture polystyrene (TCPS) and poly-l-lactic acid (PLLA). RESULTS: The fastest proliferation was seen on gelatin, followed by TCPS and PLLA, regardless of seeding density. On both TCPS and gelatin, maximal proliferation was attained at an initial seeding density of 1000 cells/cm(2). At seeding densities above and below 1000 cells/cm(2), the proliferation rate decreased sharply. On PLLA, there was a decrease in cell numbers over 7 days of culture, below a certain threshold seeding density (c. 2500-3000 cells/cm(2)), which meant that some of the cells were dying off rather than proliferating. Above this threshold seeding density, HUVEC displayed slow proliferation. Subsequently, quantitative real-time polymerase chain reaction (RT-qPCR) analysis of eight gene markers associated with adhesion and endothelial functionality (VEGF-A, integrin-α5, VWF, ICAM1, ICAM2, VE-cadherin, endoglin and PECAM1) was carried out on HUVEC seeded at varying densities on the three substrata. A significant downregulation of gene expression was observed at an ultralow cell-seeding density of 100 cells/cm(2). This was accompanied by an extremely slow proliferation rate, probably because of an acute lack of intercellular contacts and paracrine signaling. CONCLUSION: Hence, this study demonstrates that seeding density has a profound effect on the proliferation and gene expression profile of endothelial cells seeded on different biomaterial surfaces.

Adhesion, proliferation, and gene expression profile of human umbilical vein endothelial cells cultured on bilayered polyelectrolyte coatings composed of glycosaminoglycans.

This study characterized human umbilical vein endothelial cell (HUVEC) adhesion, proliferation, and gene expression on bilayered polyelectrolyte coatings composed of an outermost layer of glycosaminoglycans (hyaluronan, heparin, or chondroitin sulfate), with an underlying layer of poly-L-lysine or chitosan. The proportion of cells that adhered to the various polyelectrolyte coatings after 1 and 2 h incubations was quantified by the WST-8 assay. Interchanging poly-L-lysine with chitosan resulted in significant differences in cellular adhesion to the outermost glycosaminoglycan layer after 1 h, but these differences became insignificant after 2 h. The proliferation of HUVEC on the various bilayered polyelectrolyte coatings over 10 days was characterized using the WST-8 assay. Regardless of whether the underlying layer was poly-L-lysine or chitosan, HUVEC proliferation on the hyaluronan outermost layer was significantly less than on heparin or chondroitin sulfate. Additionally, it was observed that there was more proliferation with poly-L-lysine as the underlying layer, compared to chitosan. Subsequently, real-time polymerase chain reaction was used to analyze the expression of seven genes related to adhesion, migration, and endothelial function (VWF, VEGFR, VEGFA, endoglin, integrin-α5, ICAM1, and ICAM2) by HUVEC cultured on the various bilayered polyelectrolyte coatings for 3 days. With poly-L-lysine as the underlying layer, biologically significant differences (greater than twofold) in the expression of VWF, VEGFR, VEGFA, endoglin, and ICAM1 were observed among the three glycosaminoglycans. With chitosan as the underlying layer, all three glycosaminoglycans displayed biologically significant differences in the expression of VWF and VEGFR compared to the chitosan control. CT-HA displayed the highest level of expression of VWF, whereas expression levels of VEGFR were almost similar among the three glycosaminoglycans.