Abstract
Cystic fibrosis (CF) is a chronic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes for a channel expressed at the apical surface of epithelial tissues. Defective chloride and bicarbonate secretion, arising from CFTR mutations, cause a multi-organ disease. In the airways, impaired ion transport results in a thick mucus, dehydration of the periciliar region and bacterial infections. Over the last years, basic research has sustained a great effort to identify therapies that are able to correct defective CFTR. For this purpose, in vitro cell models have played a key role in the study of mechanisms of the disease and to assess CFTR modulator therapies. Cultures of human primary bronchial epithelia are considered a physiologically relevant disease model due to their ability to maintain most of the morphological and functional characteristics of the airway epithelium in vivo. Despite their value, these cells are limited by the availability of human lung tissue and by the complexity of the culture procedure. However, primary human nasal cells can be considered as an alternative model for the study of CF pathophysiology since they are easier to obtain and recapitulate the properties of bronchial cultures. Over the years, several groups have optimized a protocol with key steps to culture and fully amplify differentiated primary airway epithelia. Our approach provides epithelia monolayers grown on porous filters, characterized by high transepithelial electrical resistance and an electrical potential difference. These parameters are required to perform electrophysiological experiments devoted to the study of ion transport mechanisms in airway epithelia. The aim of this study was to describe different methods to expand and differentiate isolated cells into fully polarized monolayers of airway epithelium, in order to provide an optimized protocol to support physiopathology analysis and to evaluate therapeutic strategies.
Highlights
Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the gene coding for the protein cystic fibrosis transmembrane conductance regulator (CFTR)
CFTR is a chloride channel expressed at the apical surface of secretory epithelia where it plays an important role in salt and fluid homeostasis (Saint-Criq and Gray, 2017)
Isolated cells are cultured and expanded with a high proliferation rate using a proliferative serum-free medium. This step is followed by epithelial cell differentiation on permeable supports, whose ion transport properties can be evaluated with electrophysiological techniques (Galietta et al, 1998; Scudieri et al, 2012)
Summary
Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the gene coding for the protein cystic fibrosis transmembrane conductance regulator (CFTR). The aim of this article was to present a detailed protocol, optimized in our laboratory, for culture and differentiation of airway epithelia This method results in large-scale production of isolated HBEC and HNEC and fully differentiated epithelia that exhibit the morphological and functional defects of CF airways. These cell models are very useful for improving our knowledge about physiopathology mechanisms involved in CF and to support therapeutics strategies. Isolated cells are cultured and expanded with a high proliferation rate using a proliferative serum-free medium This step is followed by epithelial cell differentiation on permeable supports, whose ion transport properties can be evaluated with electrophysiological techniques (Galietta et al, 1998; Scudieri et al, 2012). Rock inhibitor: Y-27632 2HCl (5 μM, Sigma #SCM075). Proliferative serum-free medium: See Table 1 and
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