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J. Lomako, W.M. Lomako, C.A. Carraway, K.L. Carraway; The Ultrastructure of Cultivated Corneal Epithelium Cells From Rat Cornea Explants . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1339.
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Purpose: To establish and characterize corneal epithelium primary cell cultures for the study of sialomucin Muc4 and its tyrosine kinase receptor ErbB2 and their role in corneal epithelium function. Methods: Corneal epithelium was dissected from rat eyes along the corneal-limbal border and divided on six pieces. Explants were cultivated either on Matrigel-coated plates or on polyethylene terephtalate (PET) membrane inserts. All cultures were submerged in DMEM/F12 medium with Gluta Max, sodium pyruvate, pyridoxine.HCl, sodium bicarbonate supplemented with FBS, ITS, EGF, hydrocortisone, penicillin, streptomycin, kanamycin, gentamycin, fungizone and DMSO. The morphology of the cultured corneal epithelium on PET membrane inserts was examined by transmission (TEM) and scanning (SEM) electron microscopy. Proliferation and epithelial character of the cells were monitored by immunofluorescence with specific antibodies. Immunoblots were used to inspect expression of Muc4 and ErbB2. Results: The cells started to grow from the explants placed on Matrigel or on the PET membrane after 2 to 4 days in culture forming 4 to 6 cell-layer sheets of well-stratified epithelium. Based on immunofluorescence staining with antibody against cytokeratin 3 the sheets were composed of corneal epithelial cells. Staining with anti-BrdU and anti-smooth muscle actin suggested that the sheets were formed by migration, proliferation and differentiation processes. TEM and SEM inspections showed the presence of microvilli at the apical surface of superficial cells and in very high amounts in cells undergoing desquamation. Desquamating cells expressed significant amounts of Muc4 and ErbB2. Conclusions: The cultured cell-sheets on PET membranes and Matrigel provide a model system similar to the normal corneal epithelium for studying the roles of Muc4 end ErbB2 in corneal epithelium behavior and function in normal and pathological states.
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