Evaluating Cellular Effects of PET Microplastics in 2D/3D Models: Methodological Considerations of Reagent Interference

Despite the widespread environmental contamination by micro- and nanoplastics (MNPs), the cellular toxicity of polyethylene terephthalate (PET) particles is poorly understood. Here, we assessed pristine PET-Microplastic particles (MPs) (diameter: 1.24 ± 0.52 µm) and fluorescently labeled PET-MPs (Fluo-PET, diameter: 1.25 ± 0.97 µm) in both 2D (monolayer cultures) and 3D models (multicellular spheroids) across different mammalian cell types. PET exposure caused dose- and cell type–dependent reductions in cell viability at concentrations of 2.5–5 µg/ml, with uptake rates of up to 16%. In 3D models, optical photothermal infrared spectroscopy (O-PTIR) enabled the detection and biodistribution analysis of non-labeled particles, highlighting its value as a polymer-specific, label-free technique. Short-term exposure (10 min – 120 min) induced rapid, concentration-dependent ROS generation across all cell types, while reactive nitrogen species (RNS) levels remained unchanged. Due to numerous potential interferences, particular attention was given to identifying artifacts caused by MP interactions with conventional assays, including plastic-plate interferences due to MP adsorption during spheroid formation, distortions in fluorescence-based assays due to light scattering, dye adsorption, or surfactant effects, all of which can yield false-positive or false-negative results if appropriate controls are not implemented. Our findings emphasize the importance of accounting for such artifacts and interferences, and the need to report and discuss these effects explicitly. In summary, PET particles induce significant cytotoxicity and oxidative stress, which is confirmed after correction for experimental artifacts.