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Gary Hin-Fai Yam, Ting Zhang, Andri K Riau, Roger Beuerman, Donald Tan, Jodhbir Mehta; The Effect of Amniotic Membrane De-epithelialization Method on its Biological Properties and Ability to Promote Limbal Epithelial Cell Culture. Invest. Ophthalmol. Vis. Sci. 2013;54(15):553.
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© ARVO (1962-2015); The Authors (2016-present)
To characterize the de-epithelialized human amniotic membrane (HAM) and compare cell attachment and proliferation efficiencies.
HAM was de-epithelialized by 20% ethanol (AHAM), 1.2 U/ml Dispase (DHAM), 0.02% EDTA (EHAM), 0.25% trypsin-EDTA (THAM) and 5M urea (UHAM), respectively, followed by gentle scrapping with a #15 blade. Surface topology, extracellular matrix (ECM) and growth factor content were characterized and compared to intact HAM by electron microscopies (EM), atomic force microscopy (AFM), immunohistochemistry and western blotting. Primary human limbal epithelial cells (LEC) attachment and proliferation efficiencies were assayed. Statistical significance was calculated by SPSS and Fisher’s Least Significant Difference test.
EHAM, THAM and UHAM had intact basal lamina and smooth basement membrane surface shown under transmission and scanning EM and AFM. Cell remnants stayed on AHAM. Disrupted basement membrane and stroma was found in DHAM. Immunostaining intensity quantification and hierarchical clustering revealed that ECM composition of EHAM and UHAM resembled to intact HAM. In contrast, DHAM and THAM had drastic loss of ECM and growth factor content. LEC attachment efficiency at 24 hours post-seeding was the highest in EHAM (51% as on conventional culture surface), followed by UHAM and AHAM. However, cell proliferation indices at day 10 of culture were similar among different HAM substrates, suggesting repair of ECM and basement membrane by growing epithelial cells.
Urea denudation preserved the basement membrane integrity, ECM and growth factor composition, and had higher cell attachment and proliferation efficiencies. With its short processing time, urea treatment offers a novel alternative for HAM de-epithelialization.
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