期刊
GRAEFES ARCHIVE FOR CLINICAL AND EXPERIMENTAL OPHTHALMOLOGY
卷 245, 期 1, 页码 123-134出版社
SPRINGER
DOI: 10.1007/s00417-005-0095-3
关键词
amniotic membrane; limbal epithelial cell culture; cell suspension
资金
- Wellcome Trust Funding Source: Medline
Background: 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. Methods: 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. Results: 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 formed a monolayer.The culture on denuded AM had significantly (P < 0.001) more desmosomal junctions as well as significantly (P < 0.001) more junctional attachments to the carrier than the intact culture. In addition, the intercellular spaces between cells cultivated on denuded AM were significantly (P < 0.001) smaller than those between cells grown on the intact substrate. In cultures on both denuded and intact AM, the basement membrane zone displayed a positive staining for collagen VII, integrins alpha-6 and beta-4 and laminin 5. Conclusions: We successfully cultivated well-stratified and -differentiated limbal cells on denuded AM, while on the intact AM limbal cells failed to stratify and in places formed only a monolayer of cells. The limbal cells cultivated on denuded AM were well attached to the AM stroma and were morphologically superior to the limbal epithelium cultivated on intact AM. We conclude that for purposes of transplantation of differentiated epithelial sheets, denuded AM is probably the more practical carrier for human limbal epithelial cell cultures when using our cell-suspension culture system.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据