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Amanda J. Vernon, Stefan Schrader, Julie T. Daniels; The Potential Of Non-animal Derived Culture Components In Ex Vivo Expansion Of Human Limbal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1819.
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© ARVO (1962-2015); The Authors (2016-present)
Cultured limbal epithelial stem cell (LESC) therapy has been used to treat blinding ocular surface disease associated with LESC failure. Graft production methods are not optimal, removing animal-derived products from the culture system would improve graft safety. This study investigates limbal epithelial cell (LEC) expansion in media, supplemented with human serum (HS) as an alternative to animal-derived foetal calf serum (FCS). In addition LECs were cultured with human MRC5 foetal lung fibroblasts in order to compare their efficacy to gold-standard mouse 3T3 fibroblasts.
LECs were isolated from allogeneic cornea-scleral rims using enzymatic digestion and mechanical scraping. LECs were cultured with MMC growth arrested 3T3 feeder cells with the addition of corneal epithelial culture media (CECM), supplemented with either 10% HS or FCS. LEC characteristics were examined by light microscopy and colony forming efficiency (CFE) assay. The expression of putative LESC and differentiation markers (p63α and CK3) were determined using immunocytochemistry. Further LEC cultures were cultured in the presence of MMC growth arrested human MRC5 or mouse 3T3 fibroblasts. LEC characteristics were examined by light microscopy and CFE.
LECs cultured with HS produced large, round, smooth colonies indicative of colonies formed by poorly differentiated cells. CFE indicated HS maintained a population of LECs with proliferative capacity. Light microscopy showed small cells with typical epithelial-like cobblestone morphology exhibiting a large nuclear to cytoplasmic (N/C) ratio. HS-cultured LECs expressed both p63α and CK3. LECs expanded on human MRC5 fibroblasts maintained a population of LECs with proliferative capacity. Light microscopy showed small cells with typical epithelial-like cobblestone morphology exhibiting a large N/C ratio.
LECs maintained a poorly differentiated phenotype and proliferative capacity when cultured in HS, or when expanded on human MRC5 fibroblasts. Further validation is required to use these components within a clinical setting to ensure these components are not only safe but also effective; therefore this study is a step forward in improving such cell therapy products to patients.
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