The Analysis of Human Limbal Epithelial Cells Cultured on Several Extracellular Matrix Components

Abstract

Abstract: : Purpose: The extracellular environments, and in particular the basement membrane zones, of the limbal and corneal epithelium vary considerably. Because of this, we investigated the culture of limbal epithelial cells on several extracellular matrix (ECM) components. Methods: 3 adult human limbal rings (donor ages 74–80 years) donated for research were obtained from UK Transplant. Limbal epithelial cells, retrieved from these rings by trypsinisation, were co–cultured with mitotically inactivated mouse 3T3–J2 fibroblasts. Prior to confluence of these primary limbal epithelial cultures, the 3T3 fibroblasts were removed, and the cultured limbal epithelial cells were subcultured onto plates coated with collagen 1, collagen 4, fibronectin, laminin, or Matrigel^TM using 3T3–J2 fibroblast conditioned medium, and also onto 3T3 fibroblasts for comparison. These cultures were then investigated using the following parameters – culture morphology by phase contrast microscopy; p63 localisation in limbal epithelial colonies using fluorescent immunohistochemistry; and cytokeratin (CK) 3/12, CK19 and p63 expression by flow cytometry. Results:Limbal epithelial cells established and formed colonies on the ECM components in an identical time period to the 3T3 fibroblast co–cultures (3–5 days). The morphology of colonies on the ECM components however lacked the tight circular looking appearance of the colonies in the 3T3 co–cultures. Despite this difference in morphology, p63 expression by immunohistochemistry was still observed in the colonies on the ECM components. Flow cytometric analysis revealed the following changes in ECM, as compared to 3T3, established cultures – collagen 1 (+0.03% CK3/12, +34.65% CK19, –1.24% p63); collagen 4 (+11.32 CK3/12, 35.87% CK19, +7.60% p63); fibronectin (+32.50% CK3/12, +30.08% CK19, +12.83% p63); laminin (+12.32% CK3/12, +15.21% CK19, +5.80% p63); and Matrigel^TM (+22.06% CK3/12, + 33.72% CK19, –0.14% p63). Conclusions: Although the limbal epithelial cells cultured on the various ECM components have a more differentiated appearance morphologically as compared to 3T3 co–cultures, for collagen 4, fibronectin, and laminin there is a significant increase in the expression of p63, a marker for corneal epithelial progenitors. The preservation and expansion of limbal stem cells on these three ECM components will require further investigation.

Keywords: cornea: epithelium • extracellular matrix • flow cytometry