In vitro hepatic 3D cell models and their application in genetic toxicology: A systematic review

The rapid development of new chemicals and consumer products has raised concerns about their potential genotoxic effects on human health, including DNA damage leading to serious diseases. For such new chemicals and pharmaceutical products, international regulations require genotoxicity data, initially obtained through in vitro tests, followed by in vivo experiments, if needed. Traditionally, laboratory animals have been used for this purpose, however, they are costly, ethically problematic, and often unreliable due to species differences. Therefore, innovative more accurate in vitro testing approaches are rapidly being developed to replace, refine and reduce (3R) the use of animals for experimental purposes and to improve the relevance for humans in toxicology studies. One of such innovative approaches are in vitro three-dimensional (3D) cell models, which are already being highlighted as superior alternatives to the two-dimensional (2D) cell cultures that are traditionally used as in vitro models for the safety testing of chemicals and pharmaceuticals. 3D cell models provide physiologically relevant information and more predictive data for in vivo conditions. In the review article, we provide a comprehensive overview of 3D hepatic cell models, including HepG2, HepG2/C3A, HepaRG, human primary hepatocytes, and iPSC-derived hepatocytes, and their application in the field of genotoxicology. Through a detailed literature analysis, we identified 31 studies conducted between 2007 and April 2024 that used a variety of standard methods, such as the comet assay, the micronucleus assay, and the γH2AX assay, as well as new methodological approaches, including toxicogenomics, to assess the cytotoxic and genotoxic activity of chemicals, nanoparticles and natural toxins. Based on our search, we can conclude that the use of in vitro 3D cell models for genotoxicity testing has been increasing over the years and that 3D cell models have an even greater potential for future implementation and further refinement in genetic toxicology and risk assessment.