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U.S. Schrehardt, C. Cursiefen, T. Dietrich, C. Hofmann–Rummelt, F.E. Kruse; Phenotypic Characterization of Human Limbal Epithelial Cells Expanded on Fibrin Gels . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3945.
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© ARVO (1962-2015); The Authors (2016-present)
To characterize the phenotype of human limbal epithelial cells expanded on a standardized biological substrate with regard to morphology, growth potential, and antigen expression patterns, and to evaluate their clinical application in corneal surface reconstruction.
Human corneal limbal specimens were obtained from 10 donor eyes and expanded by explant culture on fibrin gels with 3T3 feeder cells. The phenotypes of primary cultured cell sheets were evaluated by light and electron microscopy and by immunohistochemical staining with antibodies for putative stem cell markers, differentiation markers, basement membrane markers, and adhesion molecules. BrdU labeling was performed to identify the proliferative activity and presence of label–retaining cells. Subcultivated cells were further evaluated for their colony–forming efficiency (CFE). After standardization of the technique, fibrin–cultured autologous epithelial sheets were transplanted onto the corneal surface of 5 patients with limbal stem cell deficiency due to chemical burns.
Outgrowth of epithelial cells from all explants, regardless of donor age, was seen after 3–4 days on fibrin and reached confluence in 14–21 days. Light and electron microscopy demonstrated monolayers of cuboid cells containing euchromatin–rich nuclei with prominent nucleoli, variable amounts of cytoplasmic filaments, apical microvilli, and small adherens junctions but no desmosomes between adjacent cells. Cultured cells expressed vimentin, p63, EGFR, alpha–enolase, integrins beta1, alpha9 and alpha3beta1, syndecan–1, P–cadherin, partly also ABCG2, but were negative for K3/12, K5/14, K19, Cx43, and desmoglein. Hemidesmosome formation and positive labeling for integrins alpha6 and beta4 was observed after 14–15 days, whereas deposition of basement membrane material and positive labeling for type IV collagen and laminin–1 occurred after 17–20 days. BrdU–label retaining cells were identified in 2.1 ± 0.9% and colony–forming cells in 3.4 ± 1.5% of fibrin–cultured cells. After transplantation of autologous fibrin–cultured limbal cells, the patients‘ corneas showed complete epithelialization and a decrease in corneal scarring and vascularization by 2 weeks following surgery.
Fibrin–cultured cells retained characteristics similar to those of the limbal stem cell compartment in situ. Fibrin gels, therefore, represent a standardized and efficient substrate for adhesion, proliferation, and maintenance of limbal epithelial cells for successful clinical application.
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