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A rapid method for culturing guinea pig gastric mucous cell monolayers
Authors:D W Rattner  S Ito  M J Rutten  W Silen
Institution:(1) Department of Surgery, Beth Israel Hospital, 02115 Boston, Massachusetts;(2) Department of Anatomy and Cellular Biology, Harvard Medical School, 02115 Boston, Massachusetts
Abstract:Summary A method has been developed for growing confluent primary cultured monolayers of guinea pig gastric mucous cells suitable for in vitro electrophysiological, transport, and pharmacological studies. Isolated mucous cells were enriched on a one-step Percoll density gradient and plated on fibronectin-coated plastic dishes or in small cups with holes containing glutaraldehyde-fixed Vitrogen gels. These cups were designed to fit in Ussing chambers. Mucous cells attached, proliferated, and formed confluent monolayers in 3 d. The low cuboidal cells contained periodic acid Schiff-positive mucous granules that were negative by Bowie and indirect immunofluorescent staining for pepsinogen. Electron microscopy revealed polarized mucous cells with microvilli, mucous granules, microfilaments, small mitochondria, some vacuoles, and junctional complexes that excluded wheat germ agglutinin-peroxidase. No basal lamina was present. Monolayers could be maintained for over 2 wk but subcultures were not made. The cultures were virtually free of fibroblasts. Epithelial sheets produced by this simple and rapid method can be used for electrophysiological, ion transport, and pharmacological studies. This research was supported in part by National Institutes of Health grants GM7806, AM31158, AM 15681, and AM 30303.
Keywords:primary cell culture  gastric mucous cell  cultured monolayer  gastric ion secretion
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