Immortalization of human airway epithelial cells for genome editing applications

Aim In vitro air–liquid interface (ALI) cultures of human airway epithelial cells are widely used to study respiratory biology and disease, as they recapitulate key features of the pseudostratified airway epithelium and reduce reliance on animal models and repeated patient biopsies. However, the limited proliferative capacity of primary airway basal cells restricts long-term studies and genome editing. This study aimed to generate stable, immortalized human airway basal cell lines that retain differentiation capacity and support genetic manipulation. Methods Human airway epithelial basal cells from bronchial and nasal origin were immortalized by lentiviral expression of human telomerase reverse transcriptase (hTERT) followed by polyclonal expansion. A panel of cell lines derived from three independent nasal donors was established. Differentiation capacity was assessed in ALI cultures and compared with primary airway epithelial cells. Suitability for genetic manipulation was evaluated using lentiviral overexpression and CRISPR/Cas9-mediated gene knockout and knock-in strategies. Results All immortalized cell lines displayed sustained proliferative capacity and efficiently differentiated into major airway epithelial cell types, including multiciliated and secretory cells. Differentiation patterns and functional characteristics closely resembled those of primary airway epithelial cultures. The cell lines supported efficient lentiviral transduction and CRISPR/Cas9-based genome editing, enabling stable gene overexpression, targeted gene disruption, and precise genomic modification. Conclusions hTERT-immortalized human airway basal cell lines preserve key features of primary airway epithelium while enabling long-term culture and versatile genome editing. This resource provides a robust platform for airway biology research, disease modeling, and therapeutic development.