Characterizing airway epithelial cultures grown on permeable inserts with different pore densities

In vitro airway epithelial models, such as air-liquid interface (ALI) cultures, are widely used for disease modeling, therapeutic and toxicology testing, and studying epithelial-environmental interactions. However, it remains unknown whether the pore density of the permeable membrane inserts, on which primary human bronchial epithelial cells (HBEC) are grown, influences their morphological and differentiation characteristics. To investigate this, HBEC from seven donors were cultured on inserts with low (4 × 10⁶ pores/cm²) and high (1 × 10⁸ pores/cm²) pore densities and differentiated for 28 days. ALI cultures on low pore density inserts exhibited greater epithelial thickness, longer cilia, and higher nuclear density compared to those on high pore density inserts. Additionally, periodic-acid Schiff staining revealed increased mucin production in low pore density cultures. Immunohistochemistry showed significant differences in cytokeratin 5 and FOXJ1 expression between conditions, while MUC5B and β-tubulin-IV expression was unchanged. Similarly, transepithelial electrical resistance measurements indicated similar barrier integrity. Our findings indicate that pore density influences HBEC-derived ALI culture morphology and differentiation, which may contribute to variability across studies and highlight that selecting the appropriate insert is crucial for experimental consistency.