Monolayer (2D) or spheroids (3D) cell cultures for nanotoxicological studies? Comparison of cytotoxicity and cell internalization of nanoparticles

Two-dimensional (2D) cell culture monolayers are commonly used for toxicological assessments of nanomaterials. Despite their facile handling, they exhibit several constraints due to their structural and complexity differences with three-dimensional (3D) in vitro cell models, such as spheroids. Here, we conducted a comparative nanotoxicological study of fibroblasts (L929) and melanoma (B16-F10) cells, grown in 2D and 3D arrangements. The cytotoxicity, reactive oxygen species (ROS) production, genotoxicity, cell morphology complexity, and uptake of silver nanoparticles (AgNPs) and folic acid-functionalized upconversion nanoparticles (FA-UCNPs) were compared in the two culture arrangements. AgNPs cytotoxicity was higher in spheroids than in monolayer cultures. Furthermore, apoptotic cell percentages and ROS production were higher in 3D than in 2D cell cultures. More importantly, 2D cultures required twice the concentration of AgNPs than the 3D cell models to reach a considerable DNA damage index (c.a. 200). Therefore, spheroids are more sensitive to the genotoxic effects of AgNPs. FA-UCNPs exerted negligible cell toxicity in 2D and 3D cell models. Moreover, AgNPs induced disaggregation and downsizing of spheroids in a facile and concentration-dependent manner. Internalization of FA-UCNPs in spheroids was 20% higher than in the 2D cell arrangements. Collectively, our findings, demonstrated that spheroids are a more sensitive model than monolayers for the assessment of nanoparticle biocompatibility and internalization.