The effect of growth factor supplementation on corneal stromal cell phenotype in vitro using a serum-free media.

Abstract In order to expand cells quickly and in high numbers for corneal tissue engineering applications corneal stromal cells, or keratocytes, are often cultured in the presence of serum. However, keratocytes become fibroblastic when exposed to serum leading to a downregulation of corneal stromal specific markers. The purpose of this current study was to determine if corneal stromal cells, made fibroblastic by serum, could display native quiescent keratocyte characteristics when cultured under serum-free conditions supplemented by different growth factors. Markers specific to a native keratocyte phenotype such as keratocan and aldehyde dehydrogenase 3A1 (ALDH3A1) and those specific to a fibrotic phenotype such as α-smooth muscle actin (αSMA) and collagen type III were examined. Cells were cultured in monolayer, self-assembled pellets or collagen hydrogels. Growth factors known to modulate keratocyte phenotype were chosen to supplement the serum free media, specifically insulin-like growth factor 1 (IGF-1) and transforming growth factor beta 1 and 3 (Tβ1 and Tβ3). The effects of serum-free media, growth factors and culture system on cell proliferation and morphology and extracellular matrix (ECM) synthesis were evaluated. The expression of keratocyte markers was evaluated by real-time PCR, immunofluorescent staining and western blotting. In addition, cell migration was tested using scratch assays. When serum was removed from the cells they displayed a reduction in proliferation and ECM synthesis (not significant), in addition to a significant decrease in migratory capacity ( p p p p p p