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J.–A. Lim, H.–J. Kim, C.–K. Joo; Reconstruction of Corneal Epithelium Using Corneal Limbal Stem Cell on Amniotic Membrane . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2091.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To examine the characteristics of reconstructed corneal epithelium cultured on denuded amniotic membrane (AM) using corneal epithelial cells having differential proportions of limbal stem cells. Methods: Corneal epithelial cells were taken from human corneoscleral rims. After primary culture, limbal stem cells were separated from corneal epithelial cells through Hoechst dye efflux assay by flow cytometry (FACSVantage). The corneal epithelial cells having the differential proportions with 1% and 4.5% of limbal stem cell were prepared to reconstruction, respectively. Then, 3–dimensional (3D) culture for reconstruction of corneal epithelium was cultured on 12–mm insert tied denuded human AM on bottom. Three weeks containing airlifting later, reconstructed corneal epithelium was examined morphological research by immunohistochemistry, confocal microscopy, electron microscopy, western bolt, and RT–PCR. Results: Limbal stem cells were separated from corneal epithelial cells using hoechst 33342 dye and verapamil by side population (SP). The reconstruction of corneal epithelium was predominantly formed 5–7 layers on MilliporeTM membrane and amniotic membrane in vitro 3D culture system. The reconstruction efficiency was enhanced on 3D culture of corneal epithelial cells having more limbal stem cells. Moreover, the proportion of limbal stem cells in reconstruction process is very important to differentiation, proliferation, and stratification. Conclusions: The reconstructed corneal epithelium on amniotic membrane (AM) is healthier than epithelium on other membrane. Also, the reconstruction of corneal epithelium having more limbal stem cells on amniotic membrane was observed better differentiation, proliferation and stratification, when compare with other having less in the same of period and culture condition. This finding supports the important of stem cell number on AM for reconstruction and clinical approach.
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