(413) Patient-derived lung airway model to study CFTR function

Background: Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. CFTR mainly serves as an ion channel enabling chloride and bicarbonate transport in the airways. Loss or dysfunction of CFTR channel activity at the apical membrane of airway epithelial cells causes dehydration of the periciliary and mucus layers leading to impaired mucociliary clearance (MCC) and airway mucus plugging. It is crucial to develop robust CF in vitro models to understand effects of mutations on CFTR function. Current in vitro airway models rely heavily on transwell cultures that consist of an epithelial monolayer on inserts. The aim of this study was to design and develop a three-dimensional (3D) lung airway model incorporating patient-derived primary cells into an extracellular matrix (ECM) within a 3D bioprinted airway as a personalized system to monitor patient specific CFTR function.

Methods: Patient-derived primary airway epithelial cells (AECs) and submucosal gland ductal epithelial cells (SMGECs) were isolated from CF patient and non-CF control lung explants at transplantation. SMGECs and AECs were cultured at air-liquid interface (ALI) to induce differentiation into ciliated and mucous epithelial cells. CFTR function in isolated non-CF and CF SMGECs was monitored using short-circuit current assay at day 8 of ALI cultures. To mimic the human lung airway, a multilayered tubular structure was designed and fabricated by incorporating a hydrogel-based ECM mixed with human lung fibroblasts and AECs using a 3D bioprinting process. The fabricated lung airway was photo crosslinked to stabilize the printed structure to withstand cell culture conditions. Cell viability assays and scanning electron microscopy (SEM) were used to assess the organization and morphology of AECs and fibroblasts within the lung airway model.

Results: We successfully isolated and cultured patient-derived SMGECs and AECs from CF and non-CF lung explants and differentiated progenitor cells into ciliated and mucous epithelial cells in ALI cultures [Fig. 1A-C]. Mutant CFTR (ΔF508/ΔF508 SMGECs) function was restored in the presence of FDA-approved drug Trikafta [Fig. 1D]. Cell viability within the bioprinted lung airway was 92% with the fibroblasts forming interconnected network-like structures at day 7 [Fig. 1E-F]. Moreover, the patient-derived AECs cultured on the surface of the ECM were polarized and differentiated into mucous secreting cells and ciliated cells, forming an epithelial cell lining along the lumen side of the fabricated airway at day 30 [Fig. 1G-I].

Conclusions: We have successfully designed a 3D airway model system by developing protocols to isolate and culture patient-derived primary cells as well as fabricating a tubular airway structure to mimic the in vivo airway environment. The model incorporates AECs and fibroblasts that correctly organize within and at the surface of the ECM to mimic a human epithelialized airway. This model holds promise for tissue regeneration given that interconnected microporous structure within the ECM provides a suitable substrate for cell survival, growth, migration, and differentiation. CFTR function in patient-derived SMGECs differentiated in this airway model will further be tested using iodide efflux assay as a proof of concept. This 3D scaffold is anticipated to be a valuable novel approach to determine patient specific CFTR function to develop personalized approaches to treatment.

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