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A. Kobayashi, T. Yoshita, K. Sugiyama; In vivo Laser Confocal Microscopic Analysis of Human Corneal Epithelial Sheet Cultivated on Amniotic Membrane . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4976.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Recently, corneal epithelial sheet ex vivo expanded on amniotic membrane have been developed for severe ocular surface disorders. We have already reported the in vivo laser confocal microscopic findings of "simple" amniotic membrane (Kobayashi, AJO 2003,136;569–572). In this report, we used laser scanning confocal microscopy (Heiderberg Retina Tomograph II Rostock Cornea Module (HRT II–RCM)) to analyze the non–fixed human corneal epithelium cultivated on amniotic membrane. Methods:Four pieces of human corneal epithelium cultivated on amniotic membrane (epithelial sheet) were produced. Before examination, air–lifting for three days were performed. For confirmation of epithelial phenotype, antibody for K3 was used for immunohistochemical staining. Using HRT II–RCM, the non–fixed corneal epithelial sheets in the culture plate were visualized layer by layer. Results:By immunohistochemistory for K3 staining, corneal epithelial phenotype in the epithelial sheet was confirmed. In all pieces of epithelial sheet, similar images were obtained by HRT II–RCM. Superficial layer cells seemed masaic, similar to the esuperficial pithelium in the human cornea. However, clusters of spindle–shaped epithelial cells were sporadically observed in the superficial cell layer. On the other hand, the plane of the basal layer of the epithelium showed highly reflective, honey–comb shaped/meshwork images, which is smaller in size compared to the actual human corneal epithelial basal cells. In the amniotic based membrane layer, acellular structure with moderate reflectivity and strie–like images were observed. Conclusions:These results indicate that HRT II–RCM can be used to visualize non–fixed human corneal epithelial sheet cultivated on amniotic membrane. The significance of spindle–shaped superficial epithelial cells and highly reflective, honey–comb shaped/meshwork images in the epithelial basal layer plane need to be elucidated. HRT II–RCM may be useful in monitoring the epithelialization/stratification of the cells and quality evaluation of epithelial sheets before transplantation.
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