Repeated ENDS Exposure in Donor-Matched Adolescent Bronchial Co-Cultures

Abstract

The rapid uptake in use of Electronic Nicotine Delivery Systems (ENDS) among adolescents has created a critical knowledge gap regarding their cumulative, long-term health impacts. Typically, in vitro inhalation studies, including ENDS aerosols, rely on single exposure designs and immortalized or single-cell systems, which fail to reflect real-world use patterns or the multicellular architecture of the bronchial epithelium. To address this fundamental gap, we employed a repeated ENDS aerosol exposure paradigm to a primary co-culture system pairing differentiated primary human bronchial epithelial cells (dpHBECs) at the air-liquid interface with donor-matched primary human lung fibroblasts (pHLFs) from six sex-balanced adolescent donors. Co-cultures were exposed to either ENDS aerosols, a propylene glycol: vegetable glycerin (PG:VG) vehicle, and matched incubator controls, for 1 or 5 days in CelTox units, and evaluated across in vivo relevant respiratory phenotypic and transcriptomic endpoints. Repeated (5-day) exposures to ENDS aerosols produced distinct response profiles compared to single (1-day) exposures, including increased cytotoxicity, altered responses in ciliary beat frequency and Mucin 5AC (MUC5AC) secretion, and donor-specific pro-inflammatory cytokine secretion. Transcriptomic analyses revealed cell type-specific and exposure duration effects such that dpHBECs showed limited transcriptional response, whereas pHLFs displayed divergent 1-day versus 5-day signatures with a progressive shift in oxidative stress, DNA damage, fibrosis, and lung cancer pathways after repeated exposures. Together, these data demonstrate that repeated-exposures and multicellular in vitro models may better capture a cumulative response and cell type-specific biology that acute and single-cell systems may miss. Additionally, inclusion of diverse donors, such as pediatrics, is critical for capturing inter-individual variability relevant and predicting long-term health outcomes in susceptible populations. This talk will discuss the implications of these findings for characterizing long-term respiratory risk and for advancing complex in vitro models as predictive tools for inhalation toxicology.