| PURPOSE. To report the feasibility of using rabbit autologous conjunctival fibroblasts instead of 3T3 fibroblasts as feeder cells for the cultivation of transplantable oral mucosal epithelial cell sheets.METHODS. Rabbit conjunctival fibroblasts were harvested using explant culture method. The safety dosage of mitomycin C for rabbit conjunctival fibroblasts was analyzed using MTT method. Appropriate dose was selected within the safety range to suppress the proliferation of rabbit conjunctival fibroblasts. Rabbit oral mucosal epithelial cells were cultured on denuded human amniotic membrane with autologcus conjunctival fibroblasts derived feeder cells. Tissue-engineered comeal epithelium was evaluated by HE stain, scanning and transmission electron microscope.RESULTS. The safety dosage of mitomycin C to treat rabbit conjunctival fibroblast; at 37℃for 2hours was under 25μg/ml. rabbit conjunctival fibroblasts cultured in 5μg/ml mitomycin C at 37℃for 2hours showed satisfying proliferation inhibition. Rabbit oral mucosal epithelial cells could be seen growing from the edges of the explants with 4 days, and reached confluence in 8-10 days. The cultivated oral epithelial sheet had four to five layers of stratified, well-differentiated cells. Electron microscope revealed that the epithelial cells were tightly connected with numerous desmosomal junctions, and were attached to the amniotic membrane with hemidesomoseomes. The apical surface of the cells were covered with microvilli. CONCLUSIONS. Autologous rabbit conjunctival fibroblasts could be used as feeder cells for engineering corneal epithelium to avoid the use of potentially hazardous xenologic 3T3 fibroblasts. |
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