The Human Airway Cell and Tissue Core will provide clinically relevant in vitro models of human airway epithelium and physiological assays of epithelial function to CF Investigators within the Center. These services will promote basic pre-clinical studies of CF pathogenesis and treatment, and thereby support a number of funded projects. To accomplish these goals, the Core will: a) establish primary cultures of human airway epithelium (HBE) from CF and non-CF lung so that pre-clinical studies of CFTR biogenesis and function, including evaluation of pharmacotherapies for CF, can be performed in differentiated human airway epithelium. Cells derived from transplant specimens will be grown on permeable supports at an air-liquid interface to induce differentiation and recapitulate the airway surface epithelium in vitro. Routine CF gene mutation analysis will be obtained from a reference laboratory, b) perform a variety of assays for CFTR function to support mechanistic evaluations of CF pathogenesis and pre-clinical therapeutics. These determinations will include Ussing chamber measurements of transepithelial ion transport, fluorometric assays of anion permeability, patch clamp, intracellular recordings, and impedance and noise analyses to define channel properties in polarized HBE. To evaluate the regulation of airway surface liquid, the core will perform assays of water permeability and transport, airway surface liquid volume, and mucus transport in differentiated HBE, in conjunction with the Imaging Core, c) provide methods for modulating gene expression in primary airway cells. Recombinant viruses will be generated to allow reconstitution of wild-type CFTR in CF airway cells, as well as the expression of other genes of interest. Silencing RNAs will be expressed using recombinant viruses and lipid-mediated transfer methods to permit down-regulation of gene expression, d) create a frozen bank of CF and non-CF airway cells and airway tissue. Isolated airway cells and segments of dissected lung will be frozen with and without prior fixation to establish a cell and tissue bank for future use, including attempts to further our understanding of genotype-phenotype relationships and allow comparison of specific mRNA and protein expression in CF and non-CF lung tissue. This core will put us in a position to compare experimental conditions and therapeutic protocols using assays of salt and water transport and mucociliary clearance in well-differentiated airway epithelia in vitro and evaluate similar conditions using in vivo measurements in clinical studies. Pharmacological or peptide-mediated manipulation of channel biogenesis or the regulation of channel function and stability, when performed in polarized HBE cells, become part of the pre-clinical dataset supporting the advancement of a therapeutic strategy.
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