A549 and Ci-hAELVi cell lines coculture as a new human alveolar epithelium model for air-liquid interface exposure

Air pollution represents a growing threat to human health, particularly affecting the respiratory system through the inhalation of gaseous pollutants and fine particles that reach the alveolar region composed mainly of alveolar epithelial type I (AT1) and type II (AT2) cells at the air-liquid interface (ALI). To reduce reliance on animal models, physiologically relevant and experimentally accessible in vitro models are increasingly needed for inhalation toxicology. In this study, we developed and characterized a simple and robust human alveolar coculture model at ALI using commercially available cell lines, A549 (AT2 phenotype-like) and Ci-hAELVi (AT1 phenotype-like). Two ALI acclimatization periods (24 h and six days) were evaluated under incubator control and clean air exposure conditions. The coculture exhibited stable metabolic activity, controlled proliferation, and reduced variability compared to monoculture. The model expressed complementary AT1- (Podoplanin, Caveolin-1, Aquaporin-5, HTI56) and AT2 pneumocyte-associated markers (surfactant proteins and HTII280) at the gene and protein levels, resulting in a mix of both the alveolar epithelial phenotypes. Importantly, the coculture model maintained epithelial integrity and functional stability during prolonged ALI exposure for up to 72 h, exceeding the typical exposure window of monoculture system. In addition, responsiveness of coculture with ZnO aerosol and lipopolysaccharides exposure, was tested demonstrating its functional relevance. Overall, this relevant coculture model provides a valuable in vitro tool for preliminary toxicological screening.