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V. A. Shanmuganathan, H. S. Dua, A. Yeung, T. Gray, A. Hopkinson; Comparison of Different Epithelial-Denuding Techniques of Human Amniotic Membrane Used for ex vivo Ocular Surface Tissue Constructs. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5741.
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The human amniotic membrane (hAM) is used as a substrate for ex-vivo expansion of corneal epithelial cells. This typically involves processing the AM to expose the underlying basement membrane (BM). This study compared the methods used to denude the AM by examining the efficiency in removing the epithelial layer whilst preserving the underlying BM.
hAMs processed for transplantation were denuded using 3 techniques. These were treatments using  EDTA  dispase and  a novel thermolysin technique using a range of incubation times to establish an optimised incubation. Samples were examined by a combination of scanning and transmission electron microscopy (EM) and were assessed morphologically by a) the removal of epithelial layer, b) the integrity of the BM and c) damage to the underlying extra-cellular matrix (ECM). Immunofluorescence was carried out for the key BM components - types IV & VII collagen, laminin 5, integrins α6 & β4. Fresh intact hAM and non-denuded devitalized hAM were used as controls.
EDTA alone (of any incubation time) failed to effectively remove the epithelial layer and was achieved only when combined with scraping. This resulted in significant damage and removal of the BM with disorganisation of the ECM. Dispase with gentle scraping required much shorter incubation times to remove the epithelial layer but caused extensive damage to the BM integrity and protein constituents and ECM. Thermolysin allowed effective removal of the epithelial layer with only minimal disruption to the BM and ECM. Immunofluorescence with the key BM proteins supported the EM findings.
This study indicates that the commonly employed methods to denude the hAM damage and may even destroy the BM. This may have implications for the quality and standardisation of tissue constructs produced. We propose that the described optimised thermolysin technique be adopted as a standardised denudation method.
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