Abstract
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.