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CC Chen, JH Chang, J Savage, MA Shatos, E Gabison, D Kure, DT Azar; Immunohistological and Biochemical Characterization of Cultured MMP-Deficient Mouse Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1759.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To generate and characterize immortalized corneal epithelial and keratocyte cell lines from matrix metalloproteinase (MMP)-deficient mice. Methods: Mouse corneas were obtained immediately after enucleation and the epithelial and stromal layers were separated under a dissecting microscope. They were cultured to generate primary and SV40-immortalized corneal cells derived from MMP-2, -3, -7, -12 deficient (MMP-/-) and wild type C57BL/6 mice. We determined the immunoreactivity of the cell lines with the following antibodies: AE1/AE3 (for most acidic keratins and known basic keratins), J7 (for 55-kD cytokeratin), vimentin, type VIII collagen (for endothelial cells), α-smooth muscle actin (α-SMA; for myofibroblasts). The cytoskeletal organization and co-localization were evaluated using epifluorescent and confocal microscopy. In addition, cellular extracts of MMP -/- and wild type mice were used to compare their inhibitory effect on vascular endothelial cell proliferation. ANOVA and student's t test were used for statistical analysis. Results: Of the epithelial and keratocyte mouse cell lines derived from MMP-/- mice (designated as MSM-E, MSM-K, respectively) and wild type mice (designated as MSW-TE, MSW-TK, respectively), the MSM-2E, MSM-3E, MSM-7E and MSW-TE exhibited uniformly polygonal-shaped cells characteristic of stratified squamous epithelium. All MSM-E and MSW-TE cell lines expressed AE1/AE3, J7 and vimentin, but not type VIII collagen and α-SMA. In contrast, MSM-K (MSM-2K, MSM-3K, MSM-7K, MSM-12K) and MSW- TK cell lines were elongated, spindle shaped with scattered whorl formation and expressed vimentin and α-SMA. Cellular extracts of MSM-7E showed significant inhibition of calf pulmonary arterial endothelial cell proliferation in vitro (P<0.05). No significant differences in proliferation were observed between MSM-7E and MSW-TE cell lines. Conclusion: The MSM-E and MSM-K mouse cell lines exhibited in vitro characteristics of corneal epithelial cells and keratocytes, respectively. MSM-E and MSM-K cell lines may be useful in studying corneal wound healing, angiogenesis and other corneal pathologic and biologic processes.
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