Hidden cytotoxicity and mitochondrial dysfunction in 3D-printing polymers: evidence from FLEX, PETG and PC

Despite the widespread use of 3D printing, the toxicological profile of commercially available filaments remains largely uncharacterized across both consumer and scientific communities. The persistent lack of comprehensive toxicological data regarding proprietary industrial additives in standard safety sheets raises significant concerns. To address this critical knowledge gap, this in vitro study systematically evaluated the biological impact of short-term (24 h) and long-term (7 days) exposure to 16 commercially available filaments-representing 8 polymer types (PLA, PETG, CPE, PC, ABS, ASA, PP, and FLEX)-on primary human dermal fibroblasts, to simulate prolonged skin contact typical for wearable and biomedical applications. By integrating standard ISO 10993-5 viability assays with high-resolution respirometry (HRR), we identified critical instances of "hidden cytotoxicity." Our findings reveal severe, material-specific metabolic disruptions in specific commercial formulations of widely used polymers, such as the tested FLEX, PETG, PC, and PP filaments. The most substantial metabolic dysfunction was recorded in the evaluated FLEX (TPU) filament, which exhibited acute toxicity characterized by immediate and progressive biological deterioration from the onset of exposure, ultimately culminating in a critical bioenergetic impairment by day 7. In contrast, a distinct toxicological paradox emerged in the evaluated PETG, PC, and PP samples. These materials triggered stress-induced compensatory hyperproliferation, characterized by an increased cell population that masked a severe, progressive decline in single-cell metabolic activity (SCMA) and single-cell fluorescence (SCF). Conversely, the tested commercial ABS, CPE, PLA, and ASA filaments demonstrated robust cytocompatibility, maintaining stable metabolic and mitochondrial functions throughout the entire long-term (7 days) exposure period. PLA exhibited a more nuanced profile: while generally supporting high overall cell viability, it induced noticeable sub-lethal metabolic stress, which we hypothesize reflects a combined effect of proprietary additives and bioenergetic alterations linked to lactate release during polymer hydrolysis. Furthermore, a certified "medical-grade" ABS variant paradoxically induced significant metabolic dysfunction, underperforming compared to standard industrial ABS. These results demonstrate that the biological safety of a 3D-printed construct cannot be extrapolated merely from its polymer base, as cytotoxicity is hypothesized to be primarily driven by proprietary additives (e.g., plasticizers, stabilizers, and pigments) and thermal degradation by-products likely generated during the 3D printing process. We conclude that the standard ISO 10993-5 viability threshold (≥70%) is insufficiently sensitive to capture latent, sub-lethal metabolic stress, underscoring the need for the adoption of more rigorous, multi-parametric screening protocols.