Human keratocyte migration into collagen gels declines with in vitro ageing

Abstract

Although senescence in various cell types has been shown to have detrimental effects on wound repair, the effect of this phenomenon on corneal function with increasing age has yet to be elucidated. This study investigated the effect of in vitro ageing on keratocyte migration into a collagen gel matrix. The keratocyte cell strain EK1.BR was cultured to late passage and a comparison of early passage migration with that of late passage migration was carried out. Early or late passage keratocytes were seeded onto 6 collagen gels (1.75 mg ml −1) for each experiment. Gels were incubated at 37°C for 72 h, stained with calcein AM (0.5 mg ml −1) and assayed for cell migration using fluorescent microscopy. Changes in the effect of EGF on keratocyte migration with age were assessed by the addition of EGF (20 ng ml −1) to 3 of the 6 gels in each experiment. Proliferative lifespan was measured by immunocytochemical detection of Ki67 activity. This study shows for the first time that keratocyte migration, and migration in response to EGF stimulation, significantly declines with increasing age of keratocytes in culture ( P < 0.001). As keratocyte migration in response to cytokine stimulation is vital for corneal repair, the accumulation of senescent keratocytes with age may impair corneal wound healing.