Abstract Rationale Human nasal epithelial cells (HNECs) are a valuable model for studying cystic fibrosis (CF) and assessing CF transmembrane conductance regulator (CFTR) modulator therapies, as they can be noninvasively obtained from individuals with CF by nasal brushing. However, expansion and differentiation of HNECs are often constrained by the limited proliferative capacity supported by existing media. We evaluated PneumaCult™-NGEx, an optimized expansion medium designed to enhance the passaging and biobanking of primary airway epithelial cells, for its ability to improve HNEC growth and preserve their differentiation potential and CFTR function. Methods Freshly isolated HNECs from non-CF and CF donors were cultured in either PneumaCult™-Ex Plus or PneumaCult™-NGEx and serially passaged until senescence. Differentiation potential was assessed by seeding expanded basal cells onto Transwell® inserts to generate air-liquid interface (ALI) cultures using PneumaCult™-ALI Medium or embedding cells in extracellular-matrix domes to form organoids using the PneumaCult™ Airway Organoid Kit. Differentiation was characterized by morphology, hematoxylin-eosin staining, immunostaining for cell-type markers, transepithelial electrical resistance (TEER), and ciliated-cell quantification. CFTR activity was measured in ALI cultures by Ussing-chamber electrophysiology and in organoids by forskolin-induced swelling (FIS) assays following treatment with CF drugs. Results HNECs cultured in PneumaCult™-NGEx exhibited the expected cobblestone morphology and expanded for an average of 36 population doublings (PD) - an increase of 22 PDs compared to PneumaCult™-Ex Plus (n = 3 in both healthy and CF donors). At ALI, PneumaCult™-NGEx-expanded cells generated a pseudostratified, ciliated, and mucus-secreting epithelium with TEER ≥ 300 Ωxcm2. These cells retained CFTR function across multiple passages (P3 to P7), responding similarly to early-passage controls in Ussing chamber electrophysiology. CF-derived HNECs expanded in PneumaCult™-NGEx supported drug-screening applications in both ALI and organoid formats. Treating F508del-CFTR organoids with elexacaftor/tezacaftor/ivacaftor (ETI) restored FIS (33% vs 7% swelling in DMSO controls) and Ussing-chamber currents (ΔIsc = 8.2 and -20.6 µA/cm2 for forskolin activation and CFTR inhibition, respectively versus ΔIsc = 1.2 and 0 µA/cm2 in DMSO controls). Additionally, HNECs derived from a CF donor with a rare mutation (I506T/1898 + 3AG) and expanded in PneumaCult™-NGEx demonstrated robust CFTR functional rescue upon treatment with ETI in Ussing chamber assays, supporting the individual’s eligibility for government-subsidized treatment with Trikafta™. Conclusions PneumaCult™-NGEx Medium enhances HNEC expansion while maintaining their differentiation and CFTR functionality at later passages. The medium enables growing physiologically relevant ALI and organoid cultures suitable for CFTR modulator therapy screening and biobanking, facilitating studies of rare CFTR variants and personalized CF therapy development. This abstract is funded by: MITACS
Kramer et al. (Fri,) studied this question.