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A. Shiraishi, T. Kobayashi, Y. Kadota, Y. Hara, Y. Shirakata, K. Hashimoto, Y. Ohashi; Cultivation of Corneal Epithelial Cells on the Collagen Gel Containing Human Dermal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5749.
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Cultivated corneal epithelial transplantation has been introduced to treat severe stem cell deficiency, and successful results have been reported. However, there is an ethical problem that should be resolved, because zenogenous mouse 3T3 fibroblasts are generally utilized as a feeder layer in the procedures. In this study, we investigated whether 3T3 fibroblasts can be successfully replaced with the collagen gel containing human dermal fibroblasts.
Normal human skin was obtained from plastic surgery, and human dermal fibroblasts were cultured with 10% FCS/DMEM. Corneal limbal epithelial cells from human corneal button were cultured under serum free condition, and seeded onto the denuded amnion attached to the collagen gel containing dermal fibroblasts. After reaching confluency, the cells were exposed to an air-liquid interface for 7-10 days. The cultured corneal epithelial sheet was examined by light and electron microscopy. Immunohistochemical staining was performed with AE5, K14, ZO-1, occludin, claudin. The cultured corneal epithelial sheet was also constructed with rabbit corneal epithelial cells and transplanted to other albino rabbits to monitor the postoperative sequelae.
The corneal epithelial cells cultivated on the collagen gel were well stratified in five to six cell layers. Electron microscopy revealed the presence of abundant microvilli, and the formation of desmosomes and hemidesmosomes, almost consistent with the appearance of normal corneal epithelium. Immunohistochemistry confirmed the presence of AE5 in the all layers, K 14 in the basal layer, and Zo-1, occludin, claudin in the superficial layer of cultivated corneal epithelial cells. The transplanted rabbit corneal epithelial cells remained stable and clear up to 10 days after the surgery.
Well stratified corneal epithelial cell sheet was successfully cultured without using 3T3 fibroblasts as a feeder layer, providing an ethically better solution in conducting corneal epithelial cell transplantation.
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