(En face view of well-differentiated human airway cultures, cilia (pink) and cell nuclei (blue))
The Airway Cell and Tissue Core at the University of Pittsburgh CF Research Center has bene culturing primary human airway epithelium for over 20 years. The overall hypothesis guiding our work is that the core defect, impaired anion transport due to CFTR dysfunction, compromises the volume and composition of the airway surface liquir (ASL) and renders the airway vulnerable to defects in mucociliary clearance, innate immunity and inflamation. Studies performed in primary cultures of human bronchial epithelial (HBE) cells have proven to be highly predictive of clinical outcomes as demonstrated by the development of Ivacaftor and Lumacafto. Therefore, access to abundant cultures of highly differentiated HBE cultures is an essential component of CF and airway epithelial research.
Goal: Provide translationally relevant in vitro models of human airway epithelia from which to study the fundamental physiologyical mechanisms that determine airway health and disease
Our five objectives:
Establish differentiated primary cultires of bronchial, nasal, and sinus human airway epithelia
Studies of airway epithelial ion transport, ASL homeostasis, innate immunity, and therapuetic response are optimally performed in well differentiated human airway epithelia. Primary cultures providea laboratory model of human airways that has led to our current understanding of disease pathogenesis and emergered as the 'gold standard' for pre-clinical studies of CF therapetucs. As our CF research center continues to grow, the demnad for these cultures has dramatically increased over the preceding years. Moreover, the need for cells with less common CFTR mutations has simultaneously increased with the CFTR2 project initiatives. To accommodate the increased demand, the Cell core has developed refined methods to increase the yield from small amounts of primary tissue, while maintaining the ability to form highly differentiated cultires. Additionally, nasal and sinus epithelial cells are now routinely cultured to allow for comparison of epithelia from distinct sights in the respiratory tract and to provide access to rare CFTR mutations that would otherwise not be available.
Extensively characterize airway epithelia to assure quality control and increase productivity
To support the research efforts of Core users, all differentitated cells producded in the Core undergo extesnive physiological characterization with the following assays: short-circut current, ASL volume, DTPA absorption, and assessment of the degree of cilation. Novel high-throughput assays have been developed to assess ASL volume and ciliation. This data is used to assure quality control, facilitate the provision of appropriate donor codes to match investigator need, and inform investigators of the unique properties of the provided cells.
Provide methods for modulating gene expression in primary airway cells
The transduction of gene expression in differentiated HBW by non-viral methods is historically inefficient. The Core generates adenovirus and lentivirus vectors for target protein over-expression or shRNA-induced endogenous protein knockdown in HBE, to evaluate potential therapeutic targets. Dicer-substrate siRNA transfection is also available, which efficiently reduces gene expression without the time constraints of generating viral particles.
Provide access to high fidelity models to study ion transport proteins
To supplement studies performed in primary cultures of airway epithelia, the Core provides access to model systems to study ion transport proteins trafficking and activity in heterologous systems.
Maintain a frozen bank of CF and non-CF airway cells and airway tissues
Genotyped airway cells and segments of dissected lung are banked with and without prior fixation to establish a cell and tissue repository for Center investigator use. Advances in cell expansion methods permit the use of archived cells for functional assays, evaluation of therapetucs, qPCR and protein expression analysis of F508del and rarer mutations.