Effect of Sub-Atmospheric Oxygen on the Culture of Rabbit Limbal Epithelial Cells

Abstract

Purpose: To investigate the effect of sub-atmospheric oxygen tensions on the maintenance and expansion of limbal epithelial cells in vitro.Materials and Methods: Limbal epithelial cells were isolated from rabbit corneas and cultured in 21%, 14%, 8%, and 2% oxygen in a co-culture system with Mitomycin C growth arrested 3T3 fibroblasts. The colony forming efficiency, proliferative ability, and cell cycle distribution of cells cultured in these different oxygen tensions were determined, and semi-quantitative RT-PCR was used to detect expression of putative limbal epithelial stem cell markers, such as ABCG2 and p63α.Results: Of the four different oxygen tensions studied, 14% and 2% supported the highest and lowest colony forming efficiency values, respectively. A greater proportion of cells were found in S and G2/M phases of the cell cycle in primary 14% and 8% oxygen cultures compared to atmospheric controls. Differentiation was promoted at oxygen tensions of 8% and below. Cells with a differentiated phenotype and limited proliferative capacity were observed in 2% oxygen. Semi-quantitative RT-PCR analysis showed that the putative limbal epithelial stem cell markers ABCG2 and p63α were expressed in 21%, 14%, and 8% oxygen, with a trend of lower expression in 8% oxygen being observed.Conclusions: Limbal epithelial cells are sensitive to in vitro changes in oxygen tension. A sub-atmospheric oxygen tension of 14% promoted the maintenance and expansion of cells with limbal epithelial stem cell characteristics in a feeder co-culture system and is, therefore, recommended for the culture of rabbit limbal epithelial cells. This may also have relevance for the culture of human limbal epithelial stem cells for therapeutic application.