Human airway epithelial cell cultures maintained at an air-liquid interface recapitulate mucociliary differentiation found in vivo, regulating ion transport, mucin secretion and ciliated cell beating to reproduce mucociliary clearance mechanisms characterisitic of the native human mucosa. Recent progress has also enabled production of polarized human alveolar type 2 and type 1 epithelial cell cultures. These cultures are an excellent model to study normal and mutant CFTR thermostability (Project 1), CFTR and ENaC interactions in the airway (Project 2), mechanisms of CFTR and ENaC regulation in the alveoli and their coupling to the airway (Project 3), nucleotide/nucleoside regulatory systems (Project 4) and their integrated function in health and disease. A Tissue Procurement and Cell Culture Core was established at the University of North Carolina in 1984, under the auspices of the Cystic Fibrosis (CF) Foundation, to provide standardized cell cultures to CF researchers. The Core has increased its output and capabilities to meet growing research demands. It routinely makes available tissues, cells and media that are otherwise unavailable and/or prohibitively expensive if purchased from commercial suppliers. The purpose of this application is to provide lung tissue specimens and large numbers of human airway and alveolar epithelial cell cultures to PPG investigators, and to support the use of cultures in mechanistic studies. To accomplish these goals, we propose the following specific functions. 1) Tissue Procurement- Obtain airway and lung tissues from CF and non-CF humans as sources of airway and alveolar epithelial cells and for biochemical studies, in situ hybridization and immuno-histochemistry. Characterize donors with respect to diagnosis, demographics, and clinical features relevant to research uses. 2) Cell Isolation and Culture- Isolate primary and passaged primary epithelial cells for distribution to project investigators. Maintain stocks of frozen cells and prepare media and substrates to support preparation of cultures as dictated by investigator needs. 3) Genetic Mainipulation of Cell Cultures- Provide support for cutting edge over-expression and/or knockdown of molecules under study by project investigators.
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