113 Immune-enriched human lung organoids as a platform for nanotoxicity evaluations

Abstract Advances in in-vitro culture systems have stimulated an interest in more reliable testing approaches that predict nanomaterial toxicity risks and align with the 3Rs framework. We have previously established a human embryonic stem cell-derived lung organoid exposure model that utilises microinjection to deliver nanomaterials into the airspace-like lumen of organoids (Issa et al. 2024). These organoids exhibit the six major proximal (goblet, basal, club, ciliated) and distal (AECI/II) epithelial cell types of the adult lung and contain functional cells, evidenced by active ciliary beating and surfactant/mucin deposition. Here, we advanced the lung organoid model complexity by incorporating a functional immune component—human embryonic stem cell-derived macrophages and assessed its suitability as a tool for nanoplastics toxicity assessment. We evaluated the pulmonary toxicity of nanometric polystyrene nanospheres (PS, 50 to 500 nm) and Poly-Ethylene-Terephthalate fragments (PET, 50 to 200 nm) from plastic bottles. Lung organoids, with and without a macrophage component, were exposed with PS or PET for up to 7 d. Lung epithelial cells showed an increased PET uptake compared to PS, with no significant impact on organoid viability. Histological analysis revealed interaction of nanoplastics with airway cells and macrophages. Importantly, macrophages were found to capture PS/PET leading to reduced mucus production and alleviated DNA damage, which were all found in the lung organoids exposed to PS/PET in the absence of macrophages. With further validation, our lung organoid-macrophage model may not only reduce the need for rodent inhalation studies but also replace the current use of simple in vitro pulmonary models for toxicology studies.