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Alexander Ljubimov, Dhruv Sareen, Loren Ornelas, Anais Sahabian, David Hemmati, Chantelle Ghiam, William Brunken, Yaron Rabinowitz, Clive Svendsen, Mehrnoosh Saghizadeh; A NEW RAPID METHOD OF DENUDING HUMAN AMNIOTIC MEMBRANE FOR LIMBAL AND STEM CELL CULTURE. Invest. Ophthalmol. Vis. Sci. 2013;54(15):998.
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© ARVO (1962-2015); The Authors (2016-present)
Human amniotic membrane (HAM) is a common substratum for culturing limbal cells for future transplantation. Best results are obtained when amniotic epithelium is removed from HAM. Most existing methods require long treatments and leave many cells attached to HAM. The purpose was to develop a reliable method of HAM denuding with effective and fast removal of amniotic epithelium.
Fresh HAM was mechanically separated from chorion, and cryopreserved in PBS with 10% DMSO. After thawing and washing, HAM was de-epithelialized by different methods: soaking in 0.02% EDTA in PBS at 37°C for 1 hour; EDTA followed by gentle scraping with electric toothbrush; EDTA followed by scraping with n-heptanol soaked cotton tip; 125 μg/ml thermolysin in PBS at 37°C for 9 min followed by gentle mechanical scraping. Alternatively, HAM placed in CellCrownTM inserts was rubbed on the epithelial side for 10-30 seconds with cotton tip soaked in 0.5 M NaOH and immediately washed in PBS. Denuded OCT-embedded HAM was cryosectioned and immunostained for typical components of limbal basement membrane (BM), including laminin α2, γ1, and γ3 chains, α1/α2 type IV collagen, perlecan, nidogen-2, and fibronectin. NaOH-denuded HAM was used to culture human telomerase-immortalized corneal epithelial cells, limbal cells from corneoscleral rims, and induced pluripotent stem cells (iPSC) derived from limbal epithelial cells. Cultured cells were checked for putative stem cell marker expression (ΔNp63α, ABCG2, and keratins 14, 15, 17, and 19) by immunostaining.
Control HAM was positive for all BM markers except for laminin α2 chain that gave weak and inconsistent staining. HAM de-cellularization with EDTA or EDTA with n-heptanol left out many adherent epithelial cells; rubbing with toothbrush produced local tears. Thermolysin and NaOH resulted in the best cell removal with continuous staining for all BM markers tested. However, thermolysin-treated HAM became fragile and could be easily damaged during manipulation, which was not seen after NaOH denuding. Corneal epithelial cell line, limbal cells, and limbal-derived iPSC all grew well on NaOH-denuded HAM. Cultured cells, especially limbal cells were positive for putative stem cell markers.
HAM de-cellularization with NaOH results in rapid and thorough amniotic cell removal, and ensures excellent preservation of HAM structure and robust stem cell growth on denuded HAM.
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