Effect of cell density and hydrogen peroxide on vascular endothelial growth factor expression by human keratinocytes cultured on microcarrier beads

Intercellular communication between keratinocytes and fibroblasts via soluble mediators is a major regulatory pathway during tissue repair. Using human keratinocyte‐populated microcarrier beads, we examined the ability of these cells to secrete vascular endothelial growth factor (VEGF); and how this expression was altered by cell density and exposure to hydrogen peroxide (H2O2). Primary human keratinocytes were initially seeded onto standard microcarrier beads (Cytoline 1) using defined medium (KGM + supplements, Cambrex) and cultured in roller bottles. At the indicated times, aliquots of beads were removed and assayed for cell content with media assayed for VEGF content (Quantikine, R&D Systems). Keratinocyte VEGF expression was dependent on cell density when grown on microcarrier supports. Total medium VEGF levels decreased from 118 to 42 pg/mL from days 7 to 11 while cell numbers increased from 350 to 500 cells/bead. The beads remained sparsely populated despite the 43% increase in cell number. With increasing cell density, the responsiveness of the cells to 5 uM H2O2 decreased. At low cell density, H2O2 resulted in an 81% increase in VEGF expression (612 vs. 339 pg/mL) whereas at higher cell number there was only a 41% increase in VEGF expression. These results show the changes in growth factor expression by human keratinocytes as a function of cell density and oxidant challenge. (NIH grant R41 GM064064)