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MAN-IL HUH, Eun-Jin Yeo, Jun Hun Lee, Hong-Kyun Kim; Generation of engineered human corneal lenticule with epithelium for selective allograft. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1410. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The aim of this study was to evaluate utility value of human corneal lenticule produced after refractive surgery with RelExTM (Carl Zeiss). We investigated whether the corneal lenticule is suitable for a scaffold which reconstituting cornea including epithelium. In this study, four decellularization protocols were evaluated to determine suitability as the scaffold, and we tested decellularized lenticules to be useful for multilayered epithelium sheet for selective allograft.
To apply corneal lenticule to selective allograft, we made corneal lenticule decellularized by 0.25% trypsin-EDTA, 0.1 % SDS, or 0.1 % Triton X-100 in hypotonic Tris buffer (10mM, pH 7.6). And then remained nucleic acid and cell morphology were visualized by DAPI and vimentin staining. Furthermore, the lenticules were cryosectioned with 10 mm thickness, and images were captured by a fluorescence microscope. To test attachment of epithelial cells on the lenticule, the lenticules were coated with various extracellular matrices (ECM) composing basement membrane, such as type I collagen, type IV collagen, or laminin. We used CnT-20 media for confluent monolayer culture of epithelial cells on lenticules, and CnT-30 media containing hormonal additives were used for making multilayered epithelium. The epithelial sheets were analyzed by histological methods.
Histological analysis of the decellularized corneal lenticules showed that trypsin-EDTA treated group is completely acellular and has not detected structure protein such as vimentin differently with other groups. Monolayer culture of epithelial cells showed that confluent culture and epithelial marker expressions are achieved by using CnT-20 media. Results of immunohistochemistry showed that multilayered epithelium on the lenticule is composed of cells expressing marker such as CK15, and CK3/12 and that is similar with result on amniotic membrane and fibrin gel.
The lenticule from human corneal stroma was successfully decellularized by 0.25% trypsin-EDTA in hypotonic Tris buffer. And generation of multilayered epithelial sheet using the lenticule was successfully achieved by using CnT-20 and CnT30 containing hormonal additives. Collectively, data show that decellularized lenticule has potential for use in selective allograft to change epithelium and can be employed as a scaffold.
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