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B. Chen, S. Mi, B. Wright, C. J. Connon; Differentiation Status of Bovine Limbal Epithelial Cells Cultured on Intact and Denuded Amniotic Membrane Before and After Air-Lifting. Invest. Ophthalmol. Vis. Sci. 2010;51(13):358.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate differences in limbal epithelial cell differentiation when expanded upon intact (amniotic epithelial cells remaining) and denuded human amniotic membrane, a commonly used substrate in ophthalmic surgery for corneal stem cell transplantation.
Animal and human tissue were handled in accordance with the statement for the use of animals in ophthalmic and visual research and declaration ofHelsinki. Ex vivo expansion of the limbal epithelial cells, in supplemented media (50µg/ml antibiotics, 5% FBS, 0.5% dimethyl sulfoxide, 2ng/ml human Epidermal Growth Factor, 5µg/ml insulin, B27 supplement medium) at 37°C, 5% CO2, continued for 2 weeks followed by 1 week under ‘air-lifting’ condition. Before and after air-lifting the differentiated (K3/K12 positive) and undifferentiated (K14 positive) cells were examined by immunohistochemistry, Western blotting and real time PCR.
Limbal epithelial cells expanded upon amniotic membrane formed 4-6 stratified layers, both on intact and denuded amniotic membrane (n>6). On denuded amniotic membrane the proportion of differentiated cells remained unaltered following air-lifting, i.e. the level of K3 protein and K12 mRNA were similar. However, within cells grown on intact amniotic membrane the number of differentiated cells increased significantly following air-lifting (>200 fold in K12 mRNA level) in marked contrast cells grown on denuded amniotic membrane
AM preparation can markedly influence a measured degree of differentiation within limbal epithelia cells following airlifting.
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