43 Development of an animal-product free <i>in vitro</i> lung model for nanomaterial hazard assessment

Abstract in vitro lung models are essential for evaluating the hazard potential of airborne particles and nanomaterials. However, most established airway and alveolar culture systems depend on animal-derived components such as foetal bovine serum (FBS) for cell survival. FBS is known for being chemically undefined which introduces biological variability, limits reproducibility and raises ethical concerns. Developing fully defined, animal-product free systems is therefore crucial for improving the reliability and ethical integrity of models for inhalation toxicology. This project aims to develop an animal-product free in vitro lung model suitable for nanomaterial hazard assessment. The immortalized human alveolar epithelial cell line hAELVi and THP-1 human monocytic cells will be gradually adapted from FBS-supplemented media to the chemically defined, animal-product free Oredsson Universal Replacement (OUR) medium, a formulation recently published (2024). Cells will be monitored throughout the adaptation process. Viability will be assessed using Trypan Blue exclusion and Alamar Blue assays, while immunofluorescent staining will be employed to visualise morphological and phenotypic changes. Successful adaptation to the chemically defined OUR medium will enable the use of these cells in the next phase of model development. This involves the fabrication of electrospun membranes that mimic the structure and mechanical properties of the lung extracellular matrix. These membranes will provide a substrate to support the co-culture of epithelial and immune cells under air-liquid interface conditions, forming the foundation of a fully animal-free in vitro lung model suitable for nanomaterial hazard assessment. Overall, this work represents a step toward the development of more reliable, reproducible and ethically responsible approaches in inhalation toxicology.