Culture of conducting airway epithelial cells in serum-free medium

Abstract

Reproducible techniques for the isolation and culture of conducting airway epithelial cells from various species including human are described. Tissues recovered after necropsy or surgery are treated with 0.1% protease at 4°C overnight. The epithelial lining cells are liberated from adventitia by flushing with ice-cold minimal essential medium containing 10% fetal bovine serum. The resulting suspension of single cells and cell clusters is centrifuged and then plated on type 1 collagen gel substratum in a serum-free F12 medium supplemented with insulin, transferrin, epidermal growth factor, cholera toxin, hydrocortisone (or dexamethasone), bovine hypothalamus extract, and retinol. Increased calcium concentration in the medium to 1 to 3 mM augments in vitro mucous cell differentiation. Both a squamouslike and mucociliary differentiation of the cultured epithelium can be identified by biochemical, immunohistologic, and morphologic means. The polarity of epithelial cell cultures is promoted by use of biphasic culture methods, in which epithelial cells are fed basally and are in direct contact with the air phase.