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T. Henkel, M. Valtink, V. Zubaty, M. Kasper, R.H. W. Funk, K. Engelmann; Growth of Limbal Corneal Epithelial Cells From Organ–Cultured Corneoscleral Remnants After Keratoplasty on Amniotic Membrane . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3597.
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© ARVO (1962-2015); The Authors (2016-present)
To develop and evaluate a method to produce limbal corneal epithelial cell–amniotic membrane allografts from organ–cultured corneoscleral remnants after keratoplasty.
Human limbal corneal epithelial cells from 38 different donors were cultured on cryopreserved human amniotic membrane using cell suspensions or in vitro explant technique. Outgrowth was examined by light–, fluorescent– and transmission electron microscopy in culture and in histological sections. Phenotype was assessed by immunohistochemical staining using antibodies against CK3/12, Cx43 and p63. Immunostaining using antibodies against aquaporins 5 and 9 and against hCG/LH–receptor was used to distinguish amniotic from limbal epithelial cells. Statistical analysis was employed to determine correlation between outgrowth of cells and donor age, post mortem time and organ cultivation duration before experiment.
Mean donor age was 58.4±17.3 years (14–83), mean post mortem time was 38.3±24.3 h (9.5–93.5), and mean organ cultivation duration was 26.9±20.6 d (7–90). Overall outgrowth rate was 29.4%. Increasing donor age, post mortem time and organ cultivation duration had a negative influence on outgrowth: Second order weighted multiple polynomial regression analysis revealed a linear correlation between outgrowth and donor age (p=0.0289) and organ cultivation duration (p=0.0006), and a quadratic correlation between outgrowth and post mortem time (p=0.0184). The in vitro explant technique was clearly superior to cell suspensions. After 6.5 weeks on average the explanted limbal corneal epithelial cells formed a confluent, multilayered epithelium on intact amniotic membrane. Positive staining of CK3/12, Cx43 (all layers) and p63 (only basal layer) verified the corneal epithelial phenotype of the outgrown cells. Aquaporins 5 and 9 and hCG/LH–receptor were expressed on both, limbal corneal epithelium and amniotic epithelium, and could not be used to discriminate the cell types.
Explant technique is a simple, reproducible method to produce limbal corneal epithelial cell–amniotic membrane allografts and can be implemented into clinical routine. Organ cultivation duration has the widest influence on successful outgrowth.
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