Establishing an air-liquid interface system for exposure of mouse laryngeal epithelial cells to cigarette smoke.

OBJECTIVE

The laryngeal epithelium is one of the first lines of defense against inhaled insults, including cigarette smoke (CS). However, a lack of suitable in vitro models has limited mechanistic studies of laryngeal epithelial injury and disease pathogenesis in response to CS. The purpose of this investigation was to establish an air-liquid interface (ALI) system for modeling airborne CS exposure in laryngeal epithelial cells.

METHODS

Primary mouse laryngeal epithelial cells were harvested, cultured, and transitioned from submerged to ALI conditions to induce epithelial differentiation. ALI cultures were characterized by electrophysiological evaluation of transepithelial resistance (TEER), histology, and immunofluorescent staining and quantification for epithelial markers and compared with native laryngeal tissue. ALI cultures were exposed to increasing doses of CS during differentiation, and epithelial barrier integrity was assessed by evaluating cytotoxicity, TEER, and structure.

RESULTS

Submerged laryngeal epithelial cultures were primarily proliferating basal cells. Differentiated ALI cultures demonstrated progressive epithelial maturation, with formation of a multilayered structure and high TEER, indicative of robust barrier integrity. Epithelial marker localization in ALI cultures closely resembled that of native mouse vocal fold epithelium. CS exposure disrupted epithelial barrier integrity in a dose-dependent manner, including increased cytotoxicity and reductions in TEER. Moderate exposure permitted some recovery of function and structure, underscoring the intrinsic resilience of laryngeal epithelial cells.

CONCLUSION

This investigation establishes a physiologically relevant in vitro model system that recapitulates key features of native laryngeal epithelium and provides a robust platform for investigating the cellular and molecular pathways governing CS-induced epithelial injury and repair.