In experimental toxicology, there is an ongoing shift towards increased use of in vitro models in compliance with the 3 Rs to reduce, replace and refine animal experiment. The importance of developing new, advanced in vitro models that, compared to animal studies, will lower costs and time for hazard characterization and risk assessment but still provide reliable results, is stressed in the regulations made by the EU Registration, Evaluation, Authorization and restriction of Chemicals (REACH). In vitro models are time- and cost effective and are ethically beneficial, compared to animal studies, give higher-throughput results, and allow utilization of human cells, which might reflect human effects better than rodent models. Thus, there is a need for development of more advanced in vitro models better resembling the in vivo situation.
Standard in vitro models, which typically include cells grown in two dimensions on a flat surface, have several limitations. This cellular arrangement lacks the complexity and structural coordination as seen in vivo, resulting in limited cellular interactions and communication. In human organs and tissues, cells are often growing in three dimensions. Thus, using in vitro models where cells are arranged in a three-dimensional (3D) structure is likely to better reflect human responses and provide more realistic results to be used in human risk assessment, to prevent adverse human effects or disease of environmental exposure.
The aim of this PhD project was to establish advanced human 3D in vitro models to be applied for hazard characterization of environmental pollutants and nanomaterials, and to map underlying mechanisms of toxicity, with main focus on genotoxicity, as a crucial endpoint in all toxicity testing. The performance of the models was compared with each other and to traditional in vitro models, to find potential similarities or differences between the models.
Two different lung models were established, one representing the lower respiratory system with tricultures and one for the upper with co-cultures. For the first model, human lung epithelial cells were co-cultivated with human endothelial cells and immune cells on both sides of membrane inserts. For the latter, human bronchial cells were co-cultured with endothelial cells. The cells were cultivated at the air-liquid interphase, to better reflect real life exposure conditions, and exposure to aerosols of chemicals or nanomaterials were performed in the Vitrocell cloud exposure chamber. Endpoints investigated were genotoxicity and cytotoxicity, as well as inflammatory markers. Further, a 3D liver model was established with HepG2 human liver cells cultivated as hanging spheroids; a dense 3D spheroidal organization of cells without adhesion to a substrate, allowing increased cell-to-cell interactions and signaling. This model was also shown to be compatible with measuring cytotoxicity and genotoxicity.
This project has contributed to new knowledge on advanced in vitro lung and liver models by application of genotoxicity testing after chemical and nanomaterial exposures. The advanced models are promising 3D models for use in genotoxicity studies and can support the hazard and risk assessment of environmental compounds in compliance with the 3R´s for next generation risk assessment.
This PhD project aimed to establish advanced human in vitro models to be applied for hazard characterization of environmental pollutants and nanomaterials (NMs), in compliance with the 3Rs to reduce, refine and replace the use of animals in toxicity testing. Models focused on were lung and liver, as inhalation via air is a main exposure route for humans to NMs and environmental pollutants. After inhalation, NMs and other pollutants are often taken up into blood and systemic circulation, and distributed to other organs. Of the NMs circulating in the bloodstream, more than 30% will be sequestered by the liver, making the liver an important target organ due to its role in the metabolism. Thus, for toxicity testing, it is important with reliable human in vitro models for both lung and liver. Toxicity testing was performed in the models for silver NMs, which are relevant for human exposure.
This project has contributed to new knowledge on advanced in vitro lung and liver models by application of genotoxicity testing after chemical and nanomaterial exposures. Genotoxicity tests, well-known for use in 2D cultures, were for the first time applied on the advanced 3D models. These are important contributions to the development of advanced models as part of new advanced methodologies (NAMs). In future, NAMs may replace (some of) the use of in vivo experimentation. The advanced models are promising 3D models for use in genotoxicity studies and can support the hazard and risk assessment of environmental compounds in compliance with the 3R´s for next generation risk assessment (NGRA).
This project has strengthened the collaboration between research institutions, on both national and international levels.