A primary culture model of differentiated murine tracheal epithelium

The goal of this study was to develop a primary culture model of differenti ated murine tracheal epithelium. When grown on semipermeable membranes at a n air interface, dissociated murine tracheal epithelial cells formed conflu ent polarized epithelia with high transepithelial resistances (similar to 1 2 k Omega.cm(2)) that remained viable for up to 80 days. Immunohistochemist ry and light and electron microscopy demonstrated that the cells were epith elial in nature (cytokeratin positive, vimentin and alpha-smooth muscle act in negative) and differentiated to form ciliated and secretory cells from d ay 8 after seeding onward. With RT-PCR, expression of the cystic fibrosis t ransmembrane conductance regulator (Cftr) and murine beta-defensin (Defb) g enes was detected (Defb-1 was constitutively expressed, whereas Defb-2 expr ession was induced by exposure to lipopolysaccharide). Finally, Ussing cham ber experiments demonstrated an electrophysiological profile compatible wit h functional amiloride-sensitive sodium channels and cAMP-stimulated CFTR c hloride channels. These data indicate that primary cultures of murine trach eal epithelium have many characteristics similar to those of murine trachea l epithelium in vivo. This method will facilitate the establishment of prim ary cultures of airway epithelium from transgenic mouse models of human dis eases.