Additive manufacturing (AM) or industrial 3D printing uses cutting-edge technologies and materials to produce a variety of complex products. However, the effects of the unintentionally emitted AM (nano)particles (AMPs) on human cells following inhalation, require further investigations. The physicochemical characterization of the AMPs, extracted from the filter of a Laser Powder Bed Fusion (L-PBF) 3D printer of iron-based materials, disclosed their complexity, in terms of size, shape, and chemistry. Cell Painting, a high-content screening (HCS) assay, was used to detect the subtle morphological changes elicited by the AMPs at the single cell resolution. The profiling of the cell morphological phenotypes, disclosed prominent concentration-dependent effects on the cytoskeleton, mitochondria, and the membranous structures of the cell. Furthermore, lipidomics confirmed that the AMPs induced the extensive membrane remodeling in the lung epithelial and macrophage co-culture cell model. To further elucidate the biological mechanisms of action, the targeted metabolomics unveiled several inflammation-related metabolites regulating the cell response to the AMP exposure. Overall, the AMP exposure led to the internalization, oxidative stress, cytoskeleton disruption, mitochondrial activation, membrane remodeling, and metabolic reprogramming of the lung epithelial cells and macrophages. We propose the approach of integrating Cell Painting with metabolomics and lipidomics, as an advanced nanosafety methodology, increasing the ability to capture the cellular and molecular phenotypes and the relevant biological mechanisms to the (nano)particle exposure.

AIMES Center for the Advancement of Integrated Medical and Engineering Sciences at Karolinska Institutet and KTH Royal Institute of Technology SE 100 44 Stockholm Sweden

Centre for Applied Autonomous Sensor Systems Örebro University SE 701 82 Örebro Sweden

Department of Mechanical Engineering Örebro University SE 701 82 Örebro Sweden

Department of Neuroscience Karolinska Institutet SE 171 77 Stockholm Sweden

Department of Occupational and Environmental Medicine Örebro University Hospital SE 701 85 Örebro Sweden

Faculty of Medicine and Health School of Medical Sciences Örebro University SE 701 82 Örebro Sweden

Inflammatory Response and Infection Susceptibility Centre Faculty of Medicine and Health Örebro University SE 701 82 Örebro Sweden

Institute of Microbiology of the Czech Academy of Sciences 140 00 Prague Czech Republic

KTH Royal Institute of Technology Department of Chemistry Division of Surface and Corrosion Science SE 100 44 Stockholm Sweden

Man Technology Environment Research Center Örebro University SE 701 82 Örebro Sweden

Uddeholms AB SE 683 85 Hagfors Sweden

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