Human limbal epithelium expanded in a complex medium or medium with human serum: a comparison of morphology and cytokeratin expression

Abstract

Purpose: We compared aspects of ultrastructural morphology and the expression of selected cytokeratins in human limbal epithelium expanded in a complex medium (COM), which contains various recombinant growth factors, hormones, Cholera toxin and fetal bovine serum (FBS), and culture medium with human serum (HS) as the only growth supplement (HS).

Methods: Limbal biopsies retrieved from corneo-scleral rings were placed with the epithelium facing down on the basement membrane surface of samples of human amniotic membranes. The biopsies were cultured in parallel in either COM or HS at 37°C, 5% CO2, and 95% air. Culture medium was changed every 2-3 days and the epithelium was expanded for 3 weeks. Specimens were examined by light microscopy, transmission electron microscopy (TEM) and immunohistochemistry. Expression of CKs 3, 7, 12, 14, and 19 was evaluated using fluorescence microcopy, and intensity was graded on a scale from 0 (negative) to 3 (strongly positive).

Results: By TEM, cytoplasmic density and distribution of intermediate filaments showed a similar pattern in epithelial cells expanded in both types of medium. In basal cells, the filaments were loosely organized and inconspicuous apart from those associated with intercellular junctions. Clusters of linear or wavy intermediate filaments (tonofilaments) were encountered to a varying extent in the cytoplasm of cells in the supra-basal layers. By immunohistochemistry, expression of CK3 was not detected (grade 0) in sections of expanded epithelium regardless of type of medium. Both COM and HS supported the expression of CK 7, 12, 14 and 19, and grading of the samples for intensity of fluorescence revealed a similar pattern in epithelium engineered in the two types of medium. Intensity of fluorescence in sections of epithelium labeled for CK12 and CK19 was evaluated as weak (grade 1), while the intensity in sections of epithelium labeled for CK7 and CK 14 was evaluated as strongly positive (grade 3).

Conclusions: Our findings indicate that COM and HS may equally support the expression of selected CKs, suggesting similar degrees of epithelial proliferation and differentiation. Further, a difference in density of cytoplasmic intermediate filaments between basal and supra-basal cells indicates a maintained gradient for epithelial differentiation in both types of medium.

Keywords: 482 cornea: epithelium