Evaluating airway ALI model fabrication methods and comparing differentiation potential of primary and hTERT-immortalized epithelial cells

Human respiratory research encompasses a variety of fields including drug development, disease modeling, and toxicology testing. Despite the availability of traditional in vitro airway models, there is a persistent concern with their lack of physiological relevance to the human lung. Within the past decade, several advanced in vitro airway models have been constructed, which promises to provide more relevant applications in human respiratory research. However, the numerous variables associated in the generation of these advanced models can cause incomplete or inconsistent differentiation, resulting in research delays or cost overruns. In these studies, we showcase an optimal method of fabricating airway models consisting of human bronchial tracheal epithelial cells (HBECs) grown in collagen-coated 24-well plate inserts and cultured under air-liquid interface for 5 weeks. Model generation using different lots of primary HBECs as well as hTERT-immortalized HBECs were compared. In addition, various commercial media designed to promote epithelial differentiation were evaluated. Next, primary HBECs from ATCC and other commercial companies were evaluated and compared on epithelial differentiation and model morphology using optimized processes validated during the first phase of the study. All airway models were evaluated via weekly microscopy and transepithelial/transendothelial electrical resistivity measurements. Additionally, H&E and alcian blue imaging and MUC5AC and α-tubulin immunohistochemical analysis from histological samples of mature airway models were generated. These studies elucidate techniques and procedures to reliably generate 3-D airway models with consistent full epithelial differentiation across replicates using both ATCC and other commercial primary HBECs.