The Effects of Culture Confluence and Plating Cell Density on the Differentiation of Human Limbal Epithelial Cultures

Abstract

Purpose: : To determine the effect of both culture confluence and plating cell density on the differentiation of human limbal epithelial co-cultures with 3T3 fibroblasts.

Methods: : Human limbal tissue (donated for research and obtained from the UK Transplant Service) was trypsinised to obtain limbal epithelial cells. These were then cultured on mitomycin C mitotically inactivated 3T3 mouse embryonic fibroblasts. Limbal epithelial cells were plated at a density of 24,000 cells/cm² and these cultures were analysed before and after confluence. Limbal epithelial cells were also plated at lower densities of 15,000 cells/cm², 7,500 cells/cm² and 1,500 cells/cm² and these cultures were also analysed. All cultures were analysed for colony forming efficiencies (CFEs) and p63 (a marker for limbal stem cells) expression as assessed by both flow cytometry for p63 and real time RT-PCR for ΔNp63. Experiments were performed in triplicate.

Results: : The CFEs of post-confluent cultures were significantly lower than those of pre-confluent cultures (p=0.01). In addition, although the change in p63 expression following confluence as assessed by flow cytometry was not statistically significant, there was a statistically significant reduction in ΔNp63 expression following confluence as assessed by real time RT-PCR (p=0.001). Plating the cells at lower densities resulted in an increase in CFE (p=0.0001), an increase in p63 expression as assessed by flow cytometry (p<0.05), and an increase in ΔNp63 expression as assessed by real time RT-PCR (p<0.005).

Conclusions: : Culture confluence results in a loss of limbal stem cells and an increase in more differentiated cells as shown by the CFE and ΔNp63 expression data. In addition, the cell density studies show that by plating cells at reduced density (i.e. with a reduced final confluence) this results in less culture differentiation as shown by the CFE and p63 expression data. In summary, therefore, these studies show that culture confluence results in the differentiation of limbal epithelial cultures. As limbal epithelial sheet formation is used to transfer limbal stem cells clinically and sheet formation results from culture confluence, these results have important implications.

Keywords: cornea: epithelium • cornea: basic science • cornea: clinical science