A functional 3D full-thickness model for comprehending the interaction between airway epithelium and connective tissue in cystic fibrosis.
Patients with cystic fibrosis (CF) experience severe lung disease, including persistent infections, inflammation, and irreversible fibrotic remodelling of the airways. Although therapy with transmembrane conductance regulator (CFTR) protein modulators reached optimal results in terms of CFTR rescue, lung transplant remains the best line of care for patients in an advanced stage of CF. Indeed, chronic inflammation and tissue remodelling still represent stumbling blocks during treatment, and underlying mechanisms are still unclear. Nowadays, animal models are not able to replicate clinical features of the human disease and the conventional in vitro models lack a stromal compartment undergoing fibrotic remodelling. To address this gap, we show the development of a 3D full-thickness model of CF with a human bronchial epithelium differentiated on a connective airway tissue. We demonstrated that the epithelial cells not only underwent mucociliary differentiation but also migrated in the connective tissue and formed glandular structures. The presence of the connective tissue stimulated the pro-inflammatory behaviour of the epithelium, which activated the fibroblasts embedded into their own extracellular matrix (ECM). By varying the composition of the model with CF epithelial cells and a CF or healthy connective tissue, it was possible to replicate different moments of CF disease, as demonstrated by the differences in the transcriptome of the CF epithelium in the different conditions. The possibility to faithfully represent the crosstalk between epithelial and connective in CF through the full thickness model, along with inflammation and stromal activation, makes the model suitable to better understand mechanisms of disease genesis, progression, and response to therapy. Overall design: The epithelial and connective compartments of an in vitro 3D cystic fibrosis full-thickness model were analyzed by RNAseq. The full-thickness model was developed with a cystic fibrosis epithelium and a cystic fibrosis connective airway tissue (cystic fibrosis) or a cystic fibrosis epithelium and a normal connective airway tissue (hybrid). The epithelium from these full-thickness models was compared with the cystic fibrosis epithelium differentiated in the absence of the connective tissue. The FT CF EPI was the cystic fibrosis epithelium from the cystic fibrosis full-thickness model; the HFT EPI was the cystic fibrosis epithelium from the hybrid full-thickness model; The HBE was the cystic fibrosis human bronchial epithelium differentiated on porous inserts without the connective tissue; the FT CF CAT was the connective tissue from the cystic fibrosis full-thickness model; the HFT CAT was the connective tissue from the hybrid full-thickness model.