Improved method for transmission electron microscopy of ciliated cell monolayers maintained on gas-permeable membranes

A new method is described for preparing thin sections for transmission electron microscopy of ciliated respiratory epithelial cells cultivated in vitro on Teflon membranes. Hamster tracheal explant cultures were treated with collagenase to promote epithelial migration. Patches of monolayer growth occurred in the vicinity of the explants when the latter were incubated on FEP-etched Teflon membranes in Chamber/Dishes. Cells in the monolayer maintained a high degree of differentiation as evidenced by active ciliary motion. The monolayer was fixed and processed in situ. During the embedding process the chamber was supported on a solid Teflon cylinder to keep the membrane taut as it was heated to polymerize the resin. The membrane was removed and the monolayered cells were re-embedded and sectioned. Electron microscopic examination of cultures revealed clear cell profiles on a flat, even basal plane. This method provides for a normal spatial configuration of cells in a monolayer format, and should prove useful for electron microscopy studies of any cells cultured on flexible films.