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Lung-Kun Yeh, Shih-Chun Huang, Department of ophthalmology, Chang-gung memorial Hospital; Characterization of cultured corneal epithelial cells on the genipin-crosslinking Descemet membrane. Invest. Ophthalmol. Vis. Sci. 2013;54(15):541.
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Purpose: The Descement membrane is a good alternative culture carrier for cell culture. However, the Descement membrane is easily teared and rolled while peeling off from corneal tissue. Genipin, a nature crosslinking material extracted from the fruits of Gardenia jasminoides, has been widely used in many biological applications. In this study, we try to study the phenotypes of cultured epithelial cells on the genipin-crosslinker Descement membrane.
Methods: The crosslinking process between Descement membrane and genipin was performed before cell plating. The crosslinking characteristics of the genipin-fixed Descemet membrane was recorded. The mechanical strength and resistance were compared between the genipin-fixed and non-genipin-fixed Descemet membrane. The proliferation rate were study by MTT assay. The phenotype and morphological analysis of cultured cells were photographed and observed by scanning EM. The immunohistochemistry study was performed to characterize the phenotype of cultured bovine corneal epithelial cells.
Results: The MTT assay showed the proliferation rate was increased in the group of genipin-fixed Descemet membrane. The mechanical strength and resistance were increased in the group of the genipin-fixed Descemet membrane. The genipin-fixed Descemet membrane was easy handled, more smooth surface, and not easy teared. The SEM analysis showed the normal epithelial cells phenotype between the two groups. The immunohistochemistry staining showed the Keratin-3 positive , Cx-43 positive, and more P63 and Ki-67-positive cells on the genipin-fixed Descemet membrane.
Conclusions: The results demonstrated that genipin can form stable crosslinked products and is an effective crosslinking agent for Descemet membrane fixation. Therefore, these results indicated that the genipin-fixed Descemet membrane may be applied as a excellent culture carrier. The non-toxic nature crosslinker genipin may be useful for the application on the reconstruction of the ocular surface.
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