59 Development of a simple and cost-effective <i>in vitro</i> model for hazard assessment of nanomaterials in the pulmonary system

Abstract There is an increasing need for fast, simple, and high-throughput methods to quickly assess the potential toxicity of nanomaterials (NMs). In vitro systems serve as an effective tool that enable classification and grouping based on toxicity profiles. Based on the Safe and Sustainable by Design (SSbD) framework, identifying simple and cost-effective in vitro models for hazard assessment of NMs is crucial. While conventional submerged monoculture in vitro models have been commonly used to evaluate NM toxicity, their sensitivity may be limited. In contrast, co-culture models, incorporate multiple cell types, offering increased sensitivity due to their more complex cellular environment. In this study, we evaluated the sensitivity and reliability of monoculture systems (Calu-3 and A549 lung cell lines, and THP-1 differentiated macrophages) compared to co-culture systems combining these cell types, exposed to a range of concentrations of SiO2NM203 (25, 50, 100 µg/mL), Ag NM300K (2.5, 5, 25, 50 µg/mL), and ZnO NM110 (1.25, 2.5, 12.5, 25 µg/mL) nanoparticles. After the exposure, cell viability and inflammatory responses (IL-8, IL-6 and TNFα) were assessed. Our results indicated that monocultures and the Calu-3 + dTHP-1 co-culture showed a dose-dependent increase in cytotoxicity when exposed to NPs, while the A549 + dTHP-1 co-culture did not show a similar effect. Notably, significant inflammatory responses were observed in the co-culture systems, indicating that co-cultures were more sensitive than monocultures to NM exposure. In conclusion, these results suggest that co-cultures may offer enhanced sensitivity to metallic NPs compared to monoculture systems, making them a more suitable option for nanomaterial toxicity screening.