Comprehensive Protocol for Handling Human Small Airway Epithelial Cells (HSAECs) to Establish Air-Liquid Interface (ALI) Cultures With TEER-Based Barrier Integrity Assessment.

Understanding epithelial barrier function is essential for studying both its normal physiology and its role in disease, yet choosing an appropriate experimental model remains challenging. Animal models are commonly used but often suffer from interspecies differences that limit translational relevance. Human-derived cell lines offer a more suitable alternative, although establishing them often requires immortalisation strategies that involve overexpression of oncogenes, which can introduce phenotypic and functional changes. In contrast, primary cells, such as human small airway epithelial cells (HSAECs), provide a more physiologically accurate model. A critical aspect of replicating the native respiratory environment is maintaining continuous air exposure, which can be achieved through air-liquid interface (ALI) culture. This protocol provides a unified, step-by-step workflow for cultivating primary HSAECs under ALI conditions, covering the entire process from initial recovery after cryopreservation to the formation of a barrier-like layer. The protocol incorporates non-invasive methods such as transepithelial electrical resistance (TEER) measurements to monitor its integrity. While individual elements of this workflow have been described separately in different studies, a consolidated version encompassing the full workflow has not been widely available. This resource is intended for researchers with limited experience in airway epithelial culture and offers practical, clear guidance through each step of the process. Key features • Using primary HSAECs enables modelling the human respiratory barrier while avoiding limitations of immortalised or animal-derived cell lines. • ALI culture technique allows continuous air exposure, closely resembling in vivo conditions for airway epithelial cells. • TEER measurement offers a non-invasive, rapid method to assess epithelial barrier integrity without damaging the cultured cell layer. • Protocol supports barrier function studies, including but not limited to respiratory infections, allergic responses, toxicology screening, microbiome interactions, and drug delivery investigation.