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J. Moreno–Montanes, A. Fernandez–Hortelano, M. Garcia–Guzman, D. Lozano, J. Echeveste, F. Prosper; Comparison of Two Limbal Stem Cell Culture Systems as Corneal Regeneration Treatment . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3030.
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To characterize and compare the outcome of two culture systems for the ex vivo expansion of limbal stem cells (LSC).
Twenty–two donor corneal limbal epithelium rings were divided in several pieces. These pieces of explant were cultured either on plastic dishes or directly on a portion of denuded amniotic membrane (AM) spread on a culture plate insert in the presence of specific media with supplements. Cultures were incubated at 37ºC in a carbon dioxide air incubator and the medium was changed every 2 days. Ten to twenty days after first culture on plastic plates, cells were passed to denuded amniotic membrane continuing culture for up to 7–10 days. Inmunohistochemistry examinations were performed on cultivated epithelium. Expression levels of nuclear protein p63, connexin 43 and K3–K12 keratins and ABCG2 were analyzed. Limbal epitheliums cultivated directly on denuded amniotic membrane were also examined ten to twenty days after first culture, analyzing the same inmunohistochemistry markers.
Stem and progenitor cells markers such as p63 and ABCG2 expression levels were similar in both culture systems in a short to medium–term follow–up period. Culture of LSC by either of the 2 systems resulted in the appearance of a epithelial layer of CK+ cells on the amniotic membrane with normal distribution of CK3/12 and CK14 in the different layers of cells. Stem cell markers were present throughout the culture in both systems but expression decline after 20 days when LSC were cultured.
Our results suggest that both culture systems described present a similar effectiveness on the expansion and maintenance of both stem/rogenitor cells and more differentiated epithelial cells. On a long term bases interaction with the AM may revent terminal differentiation of the LSC.
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