The blood-brain barrier (BBB) protects the human brain from external aggression. Despite its great importance, very few in vitro models of the BBB reproducing its complex organization are available yet. Here we fabricated such a three-dimensional (3D) self-organized in vitro model of BBB microvasculature by means of a combination of collagen microfibers (CMF) and fibrin gel. The interconnected fibers supported human brain microvascular endothelial cell migration and the formation of a capillary-like network with a lumen diameter close to in vivo values. Fibrin, a protein involved in blood vessel repair, favored the further 3D conformation of the brain microvascular endothelial cells, astrocytes and pericytes, ensured gel cohesion and avoided shrinkage. The maturation of the BBB microvasculature network was stimulated by both the CMF and the fibrin in the hydrogel. The expression of essential tight-junction proteins, carriers and transporters was validated in regards to bidimensional simple coculture. The volume of gel drops was easily tunable to fit in 96-well plates. The cytotoxicity of D-Mannitol and its impacts on the microvascular network were evaluated, as an example of the pertinence of this 3D BBB capillary model for screening applications.

Conventional nanotoxicological assays are subjected to various interferences with nanoparticles and especially carbon nanotubes. A multiparametric flow cytometry (FCM) methodology was developed here as an alternative to quantify oxidative stress, mitochondrial impairment, and later cytotoxic and genotoxic events. The experiments were conducted on RAW264.7 macrophages, exposed for 90 min or 24 h-exposure with three types of multiwalled carbon nanotubes (MWCNTs): pristine (Nanocyl™ CNT), acid functionalized (CNTf), or annealed treatment (CNTa). An original combination of reactive oxygen species (ROS) probes allowed the simultaneous quantifications of broad-spectrum ROS, superoxide anion (O₂^•−), and hydroxyl radical (•OH). All MWCNTs types induced a slight increase of broad ROS levels regardless of earlier antioxidant catalase activity. CNTf strongly stimulated the O₂^•− production. The •OH production was downregulated for all MWCNTs due to their scavenging capacity. The latter was quantified in a cell-free system by electron paramagnetic resonance spectroscopy (EPR). Further FCM-based assessment revealed early biological damages with a mitochondrial membrane potential collapse, followed by late cytotoxicity with chromatin decondensation. The combined evaluation by FCM analysis and cell-free techniques led to a better understanding of the impacts of MWCNTs surface treatments on the oxidative stress and related biological response.