Abstract
Purpose::
To evaluate the potential of collagen vitrigel as a carrier membrane for limbal epithelial transplantation.
Methods::
Human limbal explants were cultured on collagen vitrigel membranes and collagen coated tissue culture plates (CTCPs). Upon reaching confluence, total RNA was extracted and gene expression for the following markers was analyzed by (RT)-PCR: p63 (nuclear protein: transcription factor), ABCG2 (drug resistance transporter), K3 and K12 (cornea-specific cytokeratins), connexin 43 (gap junction protein) and GAPDH (house keeping gene). p63, and ABCG2 have been considered as markers of ‘stemness’, while K3/K12 and connexin 43 may be regarded as corneal epithelial markers. Morphology of the cultivated epithelium on both the vitrigel and CTCPs was evaluated using light microscopy while ultrastructural assessments were performed using both scanning and transmission electron microscopy (SEM and TEM).
Results::
Confluent limbal explant cultures on collagen vitrigel were found to be optically transparent and light microscope examination showed a sheet of consisting of closely packed epithelial cells. Corneal epithelial differentiation markers K3, K12 and connexin 43 as well as putative stem cell markers ABCG2 and p63 were expressed in both the confluent limbal explant cultures on collagen vitrigel and on CTCP. Expression of the nuclear transcription factor, p63 appeared upregulated on the collagen vitrigel compared to the CTCP. SEM revealed a sheet of confluent epithelial cells exhibiting closely associated cell membranes, in addition to microvilli on the surface of the cells. TEM revealed desmosomes along cell junctions and hemidesmosomes were noted along the basal boundary of the cells where they were in contact with the collagen vitrigel. Other prominent ultrastructural details observed included mitochondria, endosomal vesicles and keratin fibers.
Conclusions::
Limbal explant culture on a novel material, collagen vitrigel, produced corneal epithelial sheets showing the phenotypic characteristics typical of differentiated corneal epithelium, in addition to key markers of stemness. Apparent upregulation of p63 suggests that the collagen vitrigel may function as a niche in preserving stemness of cultured cells. These findings are important as they highlight the potential of the collagen vitrigel as a carrier for use in ocular reconstructive applications.
Keywords: cornea: epithelium • cornea: basic science • gene/expression