Comparison of intact and denuded amniotic membrane as a substrate for cell-suspension culture of human limbal epithelial cells.

Graefe's Archive for Clinical and Experimental Ophthalmology = Albrecht Von Graefes Archiv Für Klinische Und Experimentelle Ophthalmologie
Noriko KoizumiShigeru Kinoshita

Abstract

We have previously developed a limbal epithelial culture system using a cell-suspension method on denuded amniotic membrane (AM). However, other workers reported that intact AM is advantageous for limbal epithelial culture in that it preserves stem cell characteristics. In this study, we cultivated human limbal epithelial cell-suspensions on both intact and denuded AM and compared the morphology and adhesion of the limbal epithelial cells on these two substrates. Human limbal epithelial cells were dissociated from donor eyes using dispase and gentle pipetting and then seeded onto intact and denuded AM as cell suspension. Limbal epithelial cells on AM were co-cultured with a MMC-treated 3T3 fibroblast feeder layer and epithelial differentiation was promoted by air lifting. Cultures were examined by light, scanning and transmission electron microscopy and differences in cellular attachments and intercellular spacing were quantified. Basement membrane complexes were examined by indirect immunofluorescence. Limbal cells grown on denuded AM were well stratified and differentiated. Cells were well attached to each other and to the basement membrane. In contrast, limbal cells cultured on intact AM failed to stratify and in places form...Continue Reading

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