Simple and high-containment lung-on-chip model for studying respiratory viral infections using human primary lung cells

Airborne respiratory viruses, such as coronaviruses and influenza, pose major threats to public health and the economy, as highlighted by the COVID-19 pandemic. Preclinical research is hindered by models that poorly mimic human tissue structure and function, often relying on immortalized cell lines and low-throughput animal studies. This limits accurate prediction of disease mechanisms, drug effects, and target suitability. Here, we report a custom-engineered, passive-flow, high-containment chip for culturing human primary bronchial epithelial cells (hPBECs) at air-liquid interface (ALI) on a large-area membrane. The dual-chamber microfluidic chip, separated by a horizontal support membrane, is enclosed in a 35 mm sealed Petri dish, enabling safe use in standard incubators without leakage or biosafety concerns. The platform supports high-resolution in-situ imaging, apical viral infection, and retrieval of cells and secretions (e.g., mucus, viral lysate) for molecular analysis. We demonstrate robust infection and replication of human coronavirus NL63 (HCoV-NL63) in differentiated hPBECs cultured up to 4 weeks at ALI. Epithelial differentiation was confirmed by immunofluorescence (e.g., ciliated cells), and infection kinetics were monitored by RT-qPCR over 7 days. The interferon-based immune response showed increased activity, with upregulation of viral response pathways (e.g., replication, inflammation, immunoregulation), and consistent activation across donors (e.g., ISG15, IFIT1). Collectively, we present a reproducible, small-scale chip model that enables high-containment in vitro studies of respiratory viruses and their effects on human airway epithelia.