Using Dispase-treated rat corneas, primary-cultures of corneal epithelium have been established which are free of contaminating cell types. Cultures were maintained for periods up to 36 days and were monitored with morphological and electrophysiological methods. Phase and scanning electron microscopy revealed a mosaic of polygonal epithelial cells which migrated over the culture plate. Actively migrating cells at the periphery of the culture presented a complex border of ruffles and filopodia. Surface specializations of the epithelial cells, i.e. microvilli, reflected those seen in vivo. Transmission electron microscopy revealed many cytological features common to the intact cornea: bundles of tonofilaments, desmosomes between adjacent cells and glycocalyx-covered microvilli on all free surfaces. These features developed over time in culture. Membrane potentials of the cultured epithelial cells could be recorded intracellularly. Therefore, it appears that pure cultures of rat corneal epithelium which maintain the morphological characteristics of their in vivo counterparts can be grown for considerable periods of time and that their electrophysiological properties can be monitored. This system offers the possibility of studying, under the controlled conditions of tissue culture, a wide variety of factors which might influence the integrity of the corneal epithelium or alter its susceptibility to disease.
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ACKNOWLEDGEMENTS This study was supported by grants from the National Institutes of Health (EY04659-Ol), the University of Minnesota Graduate School, and by an unrestricted grant to the University of Minnesota Department of Ophthalmology from Research to Prevent Blindness, Inc. The technical assistance of G. Anderson-Beckman, E. Bergquist, E. Gannon, S. Howard, S. Olson and secretarial assistance of M.J. Kuhlmey are gratefully acknowledged.