A new in vitro model of BBB and brain organoids to study the role of MG in brain development

BACKGROUND-AIM Human brain organoids are the widely used in vitro model that contribute to our knowledge about the biology and pathophysiology of nervous cells. They are 3D multicellular clusters that mimic the cytoarchitecture and the developmental pathways that occur in vivo. The most debated limitation of this in vitro model is the lack of the endothelial component. Thus, we have developed a co-culture in vitro system composed by the Blood Brain Barrier (BBB) and human brain organoids (ORGs). To investigate the strength of this in vitro model, the structure of the ORGs was assessed by evaluating the cortical layer organization. The modulation of BDNF, the crucial neurotrophic factor involved in neurodevelopment, neuroplasticity and neurosurvival, was observed. Moreover, we evaluate the role of magnesium (Mg) in neurodevelopment. METHODS ORGs are generated from iPSCs cultured with different differentiation media for 36 days. The BBB model is composed by a co-culture of human brain endothelial cells and human astrocytes in a transwell system. An inorganic and an organic Mg salt (Mg sulphate and Mg pidolate, respectively) were added to the culture media to reach the extracellular concentrations of 1 or 5 mM. The cortical layer differentiation was observed by immunofluorescence using CTIP2, TBR2 and SOX2 antibodies to detect the cortical, the subcortical neurons and the neural progenitor cells, respectively. Ultrastructural analysis was performed using light and transmission electron microscopy. BDNF was detected by ELISA. RESULTS Our data demonstrate that BDNF levels are higher in the new in vitro model mainly because of the presence of the endothelial component which is the main responsible for its secretion. Moreover, the cortical layer is more organized in the presence of the BBB. In addition, high Mg salts concentration (5 mM) ameliorates the organization of the ORGs cultured in the presence of BBB. CONCLUSIONS This study underlines the importance of the cross-talk between BBB and brain and unveils the role of Mg in brain development and in modulating the release of BDNF from the BBB.