Myo et al. Raw Data for Graphs.xlsx

Myo et al. Supplemental Materials.pdf from Myo et al., Protocol For Differentiating Primary Human Small Airway Epithelial Cells at the Air-liquid Interface. Am J Physiol: Lung Cell Mol Physiol. 2025.The air-liquid interface (ALI) culture is an important tool in pulmonary research as it models the physiological lung where the epithelium is apically exposed to air and basally to the endothelium and interstitium. While there is an abundance of research that employs primary human bronchial epithelial cells (HBECs) to study larger airways, small airway epithelial cells (SAECs) are an untapped resource in comparison. Primary SAECs are a valuable cell population as they enable the study of pathologies in the bronchioles and are also a favorable surrogate for primary alveolar epithelial cells which are invasive to collect from patients. Currently, there are limited resources on how to culture and differentiate SAECs at the ALI. Here, we provide an optimized detailed protocol to address this knowledge gap. Key culture conditions that determine the quality and uniformity of differentiated SAECs include cell passage number, pH changes caused by media exhaustion and incubator CO2, seeding density and collagen coating of the expansion flask and inserts. We also describe a FITC-dextran permeability assay to measure SAEC barrier integrity both as a pre-test to select uniform wells with strong barrier integrity before an experiment and as a post-test to evaluate treatment effects afterwards. The utility of the differentiated SAEC ALI model to ask biologically relevant questions is demonstrated by increased cytokine (IL-8, MIF, CXCL-10) production and/or epithelial damage following exposure to cigarette smoke, lipopolysaccharide (LPS) or poly(I:C).<br>