*3.3.2.1. Analyses on primary airway cells and tissues*

Characterization of *CFTR* molecular defect can partially be performed directly after biopsies of tissues that show sufficient CFTR expression. Nasal or lung airway epitheliums are optimal. These tissues are accessible by minimally invasive interventions and display an endogenous expression of CFTR transcripts and protein. Moreover, nasal and bronchial epitheliums show the same cellular composition (ciliated, goblet, columnar and immune cells) although ratios differ slightly [89]. Since quantification and detection of aberrant splicing and quantification or localization of proteins are possible in human tissues, information that they bring is crucial to assess the effect of variants and to propose a functional classification. However, this approach has its limitations and requires other functional assays to perform large-scale genotype–phenotype correlation studies. In addition, especially for nasal tissue, the low quantity of cells collected (out of 500.000 cells per brushing) only allows 'one-shot' tests and hinders mechanistic assays. Moreover, highly variable CFTR expression in heterogeneous cell types, in healthy individuals and in p.Phe508del homozygous patients has been described, varying from 0 to 100% [90]. Other genetic (see below) or environmental parameters could also modify CFTR expression levels.

### *3.3.2.2. Ex vivo culture of primary airway cells*

Culture of primary cells from CF patients can be performed with brushed nasal or bronchial cells after biopsies. Wild-type endogenous CFTR protein is expressed at the apical mem‐ brane of polarized cells. Therefore, *in vitro* monolayer culture seems no longer adapted. Obtaining polarized cells is promoted by air–liquid interface culture (ALI), proposed since the 2000s, by an *ex vivo* system of collagen-coated porous membrane on which cells are platted after a phase of monolayer amplification or directly after nasal brushing. Basal adherent cells differentiate in all airways epithelial cell types, which organize into a pseudostratified epithelium [91].

This model offers the opportunity to perform functional assays described above to determine CFTR dysfunction. Molecular defect induced by a specific mutation can be qualitatively determined if the cell donor is homozygous for this mutation.

Technical limitations such as bacterial or fungi contaminations or absence of adherence complicate culture of cells obtained from CF patients. Moreover, further studies are needed to determine if extrapolation is possible between observations in primary cells directly after brushing and after several weeks in culture media, particularly for quantitative level assess‐ ment.
