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

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01.01.2007 | Laboratory Investigation

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

verfasst von: Noriko Koizumi, Helen Rigby, Nigel J. Fullwood, Satoshi Kawasaki, Hidetoshi Tanioka, Kan Koizumi, Norbert Kociok, Antonia M. Joussen, Shigeru Kinoshita

Erschienen in: Graefe's Archive for Clinical and Experimental Ophthalmology | Ausgabe 1/2007

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Abstract

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.

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Titel: Comparison of intact and denuded amniotic membrane as a substrate for cell-suspension culture of human limbal epithelial cells
verfasst von: Noriko Koizumi
Helen Rigby
Nigel J. Fullwood
Satoshi Kawasaki
Hidetoshi Tanioka
Kan Koizumi
Norbert Kociok
Antonia M. Joussen
Shigeru Kinoshita
Publikationsdatum: 01.01.2007
Verlag: Springer-Verlag
Erschienen in: Graefe's Archive for Clinical and Experimental Ophthalmology / Ausgabe 1/2007
Print ISSN: 0721-832X
Elektronische ISSN: 1435-702X
DOI: https://doi.org/10.1007/s00417-005-0095-3

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