Preservation of structural and functional polarity in isolated epithelial cells

We describe a method to isolate epithelial cells from gallbladders of Necturus maculosus with preserved structural and functional polarity. Isolation was carried out with a mixture of collagenase and protease, with only a brief exposure to a divalent-cation-free medium. About 40% of the isolated epithelial cells had a "figure-eight" shape and retained metabolic and cell membrane integrity. Figure-eight cells display features consistent with preserved polarity for several hours, including the following: 1) the "apical" and "basolateral" membrane domains were differentially labeled by a hydrophobic fluorescent dye; 2) freeze fracture electron microscopy verified two plasma membrane domains differing in the presence of microvilli and folds and separated by tight junctions; 3) proteins such as ZO-1, NHE3, and Na(+)-K(+)-ATPase remained localized in the junctional, apical, and basolateral regions, respectively; 4) after apical surface exposure to wheat germ agglutinin, the label remained in the apical membrane after cell isolation; and 5) patch-clamp experiments demonstrated polarized expression of K+ channels. Polarity was rapidly lost after removal of extracellular Ca2+, exposure to trypsin, or ATP depletion. Therefore, this preparation allows for structural and functional studies of epithelial transport in single cells retaining the essential features present in the assembled epithelium.