A Novel Air‐Liquid Interface Exposure System for Continuous Gas Exposure of Airway Epithelial Cells: Exposure of NO 2 With Viral Co‐Challenge

ABSTRACT Most in vitro toxicological research investigating the effects of inhaled compounds employ short‐term exposures of a couple of hours, or repeated, intermittent exposures for a limited number of days with exposure free periods, whereas in reality, we are continuously exposed, albeit to variable concentrations. While recent studies have demonstrated continuous multi‐day exposure of cell models for up to 3 days, options to assess toxic effects of long‐term continuous exposure remain limited and longer exposure durations have not yet been performed. We present a versatile system that can be used for continuous exposure of air–liquid interface cultured airway epithelial cells to gases and vapors for multiple days to weeks. This setup was used for the concurrent combined exposure to a gas and the infection with a virus. Since exposure to air pollutants has been shown to alter the sensitivity to respiratory viral infections in epidemiological studies, there is a need to combine long‐term exposure to air pollutants with exposures to respiratory viruses to better understand the nature of this interaction. We used nitrogen dioxide and respiratory syncytial virus as an example to establish and demonstrate the applicability of this setup for “long‐term” exposures. We focus on the requirements for such a setup, and how the setup was designed to meet those requirements. We also discuss the limitations of our design, and its wider applicability. The application of the exposure setup to assess cellular responses (other than direct toxicity and IL‐8 release) is outside the current scope. We are the first to describe an exposure system for long‐term continuous exposure of cells to gases and vapors in combination with viral infection, thereby providing methodology for further exploration of chemical and biological effects on a cellular level.