Disease specific ion transport abnormalities have been identified in CF airways and other affected organs leading to the hypothesis that alterations in epithelial ion cells demonstrate similar abnormalities, establishing that these physiologic abnormalities are intrinsic to the epithelial cells and not secondary effects of inflammation or circulating mediators. The availability of primary CF epithelial cultures has permitted investigation of the molecular mechanisms responsible for the observed changes, but few tissues samples are available and the cells have a short survival. Therefore, CF research continues to be limited by availability of adequate quantities of research materials. This project will develop CF airway epithelial cell lines with increased growth capability and well-characterized morphologic and phenotypic properties. We will utilize the immortalizing genes of Simian Virus 40 for these studies, based on its record of producing human epithelial cell lines with differentiated properties. The constructs selected for use are deficient in the origin of replication, and include a temperature sensitive mutant of the SV40T region. These genes will be introduced by infection with hybrid adeno/SV40 viruses, infection with retroviruses, and by transfection of plasmids with electroporation and DEAE/dextran. Clones will be selected by resistance to the antibiotic, G418, and by prolonged survival. Cells will be cultured on plastic dishes, permeable collagen matrix supports, and in heterologous trachea grafts. Cells will be cryopreserved at sequential passages to provide stocks of cells lines for shipping to cystic fibrosis investigators. Cell lines with prolonged growth will be characterized by phase contrast, light, TEM, and freeze-fracture morphologic studies. The epithelial nature and expression of SV40T antigen will be confirmed with monoclonal antibody assays. The physiologic (especially ion transport) properties will be assessed with multiple techniques, including transepithelial bioelectric studies, intracellular and ion specific microelectrodes, patch clamp, and with potential sensitive fluorescent dyes. These studies are likely to produce 6 to 20 pre- or post-crisis cell lines from CF and normal airway epithelia. Aliquots of these cells lines from specific passage numbers will be made available to collaborating CF researchers during the course of the grant.
