A primary culture model of differentiated murine tracheal epithelium.
The goal of this study was to develop a primary culture model of differentiated murine tracheal epithelium. When grown on semipermeable membranes at an air interface, dissociated murine tracheal epithelial cells formed confluent polarized epithelia with high transepithelial resistances ( approximately 12 kOmega. cm(2)) that remained viable for up to 80 days. Immunohistochemistry and light and electron microscopy demonstrated that the cells were epithelial in nature (cytokeratin positive, vimentin and alpha-smooth muscle actin negative) and differentiated to form ciliated and secretory cells from day 8 after seeding onward. With RT-PCR, expression of the cystic fibrosis transmembrane conductance regulator (Cftr) and murine beta-defensin (Defb) genes was detected (Defb-1 was constitutively expressed, whereas Defb-2 expression was induced by exposure to lipopolysaccharide). Finally, Ussing chamber experiments demonstrated an electrophysiological profile compatible with functional amiloride-sensitive sodium channels and cAMP-stimulated CFTR chloride channels. These data indicate that primary cultures of murine tracheal epithelium have many characteristics similar to those of murine tracheal epithelium in vivo. This method will facilitate the establishment of primary cultures of airway epithelium from transgenic mouse models of human diseases.
Davidson
D J
DJ
Medical Research Council Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, United Kingdom.
Kilanowski
F M
FM
Randell
S H
SH
Sheppard
D N
DN
Dorin
J R
JR
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002034:Bumetanide / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D016764:Cell Polarity; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000502:physiology; D023082:Defensins / Q000235:genetics; D005260:Female; D006801:Humans; D007633:Keratins / Q000032:analysis; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D020545:Respiratory Mucosa / Q000166:cytology* / Q000502:physiology* / Q000648:ultrastructure; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014132:Trachea; D014746:Vimentin / Q000032:analysis
C505032:CFTR protein, human; D023082:Defensins; D014746:Vimentin; D002034:Bumetanide; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D007633:Keratins; D000584:Amiloride