Epithelial cells from lung: production, cultivation and characterization: usage for validation of compounds efficacy in rescuing mutant CFTR
Year: 2012
Type: dissertation
Abstract: Cystic Fibrosis (CF) is the most common lethal genetic disease among Caucasians. It is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, a cAMP-regulated chloride (Cl-) channel that functions at the apical membrane of epithelial cells. F508del is the most common disease-causing mutation occurring in ~90% of CF patients leading to a traffic/ processing defect. Lung disease is the main cause of CF mortality. The majority of studies on wt- and F508del-CFTR proteins have been conducted in heterologous, non-epithelial or non-polarized/epithelial cellular systems. However, the efficiency of CFTR processing is cell-type and polarization dependent, being endogenous epithelial CFTR maturation significantly different from those in overexpressing systems. It is, thus important to generate models of epithelial, polarized cells so as to resemble more closely the in vivo situation. Primary cultures of human bronchial epithelial (HBE) cells are therefore instrumental for studying basic and applied aspects of CF and other lung diseases. The goals of the present work were: 1) to compare two different protocols for the isolation and cultivation of primary HBE cells regarding the isolation and dissociation of the epithelial layers; 2) to characterize the established primary HBE cultures regarding epithelial differentiation and polarization; and 3) to assess suitability of HBE monolayers to investigate efficacy of small-molecule compounds in rescuing F508del-CFTR traffic. Our data showed no significantly differences between the two protocols tested. When cultured as air-liquid interface (ALI) cultures, HBE cells are able to polarize, thus closely recapitulating their physiological morphology and epithelial characteristics. Finally, we tested these cells by Western blot for the maturation of F508del-CFTR protein following treatment with different small-molecule correctors, showing the suitability of these HBE monolayers as a valuable pharmacology model for CFTR modulator drug discovery.
Author: Marta Palma
Language: English
Subfield: Pulmonary and Respiratory Medicine
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W121243863