P18-95 Development of human-based in vitro models to evaluate the neurotoxic effects of advanced materials

Currently, there is a lack of realistic human-based in vitro models to assess the neurotoxicity of advanced materials (AdMa). Specifically, nanometric AdMa may enter the body through routes like inhalation, potentially translocating to the Central Nervous System (CNS) by crossing the Blood-Brain Barrier (BBB) and reaching the brain. Once within the CNS, these materials could cause damage to brain tissue. However, the existing in vitro models do not represent the in vivo microenvironment of the human CNS. To study the neurotoxicity of AdMa in the CNS, we developed a human-based Blood-Brain Barrier (BBB) model using immortalized human cerebral endothelial cells and astrocytes. The BBB model was established in transwells with 0.4 µm in pore size to represent more closely the physiological barrier, seeding endotheliallike cells in the apical compartment, and adding astrocyte cells in the basolateral chamber to promote the formation of tight junctions. Also to evaluate the potential neurotoxic effect of AdMa, we developed a neurotoxicity co-culture model consisting of neuron-like cells and microglial cells (1:1) that represent two abundant cell populations of the brain tissue to represent the human brain.These models were designed to replicate in vivo conditions as closely as possible, incorporating the heterogeneity of different cell populations and physiological barriers, while also serving as robust yet sensitive systems to study the effects of AdMa on the CNS. To validate these models for neurotoxicity studies, the models were exposed to a range of concentrations of graphenebased AdMa (ranging from environmentally relevant levels to high concentrations: 0.005 to 50 µg/ml) for 24 hours. Cell viability (measured via Alamar Blue), oxidative stress (ROS production), inflammatory response (IL-6 and IL-8 levels), and other neurotoxicity markers were analyzed. The integrity and permeability of the BBB model were also assessed. The results showed that graphene-derived AdMa had no significant effects on BBB integrity, permeability, or cell viability after 24-hour exposure. However, mild inflammatory effects were observed, particularly at the highest concentrations tested (significant IL-6 and IL-8 production, p<0.05). Similar inflammatory responses were seen in the neurotoxicity co-culture model at the highest concentration, with no significant cytotoxic effects. Overall, the findings suggest that both the human-based BBB and neurotoxicity co-culture models are sensitive in vitro systems for assessing the acute effects of AdMa in the CNS. These results indicate the potential adverse effects of such materials and emphasize the need for developing novel human in vitro models to study the neurotoxicity of AdMa. This work was supported by the “Integrated Assessment and Advanced Characterisation of Neuro-Nanotoxicity” (iCare) project, funded by the European Commission under HORIZON-CL4-2022-DIGITAL-EMERGING-01.