3D Spheroids from Human Neurons and Astrocytes as New In Vitro Model to assess CNS cytotoxicity of Nanoparticles

Thousands of nanoproducts are already available on the market, raising concerns of extensive consumer exposure by different routes. Several studies have looked at nanoparticles (NPs) impact on the highly vulnerable nervous system. Available evidence suggests incomplete effectiveness of blood brain barrier (BBB) protection of the brain against NPs translocation. The distribution of NPs in the bloodstream raises also a particular concern of NP transfer from placenta to the fetal CNS and because BBB develops gradually in the fetal brain this type of direct exposure to NP in utero may have the most damaging consequences. Although standardized procedures for the evaluation of NPs toxicity have not yet been defined, the integration of validated in vitro-studies into safety assessment strategies is claimed by several Institutions. In our laboratories, the following two types of CNS cell spheroid (3D) models, generated by ultra low-adhesion multiple well plates, are currently under development using astrocytes and neurons of human origin for investigating CNS injury caused by neurotoxicants: (i) 3D neuronal- and astrocyte-like spheroids derived from human brain SH-SY5Y and D384 cell lines, respectively, (ii) 3D human neurospheres differentiated from mesenchymal stem cells (hMSCs) isolated and expanded from umbilical cords obtained from full-term infants. In vitro studies testing increasing concentrations (10-100 \u3bcg/ml) of Fe3O4NPs, as NP model, showed NP-induced cytotoxic effects in both D384 and SH-SY5Y spheroids, after short-term exposure. Concentration- and time-dependent cell mortality was also observed in repeated dosing schedule up to 30 days evidencing higher susceptibility of neurons than astrocytes. Cell disaggregation was also evidenced after the first week of treatment at 65 0.1\u3bcg/ml that became considerably marked at higher concentrations and over time. hMSCs (at P4) were firstly differentiated into neurons, phenotypically characterized, and then applied for generation of spheroid neurons. The use of spheroids as a model for investigating acute and chronic CNS injury induced by NPs is fairly novel. These specific cellular 3D cultures may represent good species-specific models as "near-to-in vivo" and provide with a better and more realistic predictive value, than traditional 2D cell cultures, for safety and risk assessment of new and emerging materials (i.e. NPs). (Grant from the Italian Ministries of Health, Research and Education)