The Effect of Calcium and Glucose Concentration on Corneal Epithelial Cell Lines Differentiation, Proliferation, and Focal Adhesion Expression.

Abstract

It is known that culture media composition can affect cell behavior, morphology, and gene expression. However,in the case of corneal epithelial cells, the combined role of calcium and glucose concentration in media has not previously been examined. In this study, a human immortalized corneal epithelial cell line was used to examine the effect of glucose and calcium concentrations on these cells. Cell metabolic activity, cell growth curve analysis,and relative gene and protein expression of proliferative marker extracellular related kinase (ERK) were used to study proliferation. Corneal epithelial stem cell marker NP63 and mature epithelial marker cytokeratin 3 (CK3) were analyzed by using reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry.Focal adhesions were examined by using immunocytochemistry. Cells cultured in both low-glucose, high-calcium (LG-HC) media and high-glucose, low-calcium (HG-LC) media showed similar results in both RT-PCR and immunocytochemistry analysis. NP63 expression was significantly lower and CK3 expression was higher in these groups compared with cells cultured in commercial media. NP63 and CK3 expression was also analyzed by using immunocytochemistry, which confirmed these findings. The high-glucose, high-calcium-fed cells showed the lowest expression of all markers and no gene expression of CK3. This was deemed the most unsuitable media formulation for this cell line. Focal adhesion expression was the lowest in the high-calcium, high-glucose-fed cells, with the most even distribution of this among the commercial media group. Overall, this study showed that varying glucose and calcium concentrations can have significant effects on differentiation,proliferation, focal adhesions, and metabolic activity of this cell line. It seems that an LG-HC and HG-LC formulation were interchangeable with similar proliferative and differentiation effects.