GDNF and cAMP significantly enhance in vitro blood-brain barrier integrity in a humanized tricellular transwell model

Blood-brain barrier (BBB) is a crucial membrane safeguarding neural tissue by controlling the molecular exchange between blood and the brain. However, assessing BBB permeability presents challenges for central nervous system (CNS) drug development. <i>In vitro</i> studies of BBB-permeable agents before animal testing are essential to mitigate failures. Improved <i>in vitro</i> models are needed to mimic physiologically relevant BBB integrity. Here, we established an <i>in vitro</i> human-derived triculture BBB model, coculturing hCMEC/D3 with primary astrocytes and pericytes in a transwell format. This study found that the triculture BBB model exhibited significantly higher paracellular tightness (TEER 147.6 ± 6.5 Ω × cm²) than its monoculture counterpart (106.3 ± 1.0 Ω × cm²). Additionally, BBB permeability in the triculture model was significantly lower. While GDNF and cAMP have been shown to promote BBB integrity in monoculture models, their effect in our model was previously unreported. Our study demonstrates that both GDNF and cAMP increased TEER values (around 200 Ω × cm² for each; 237.6 ± 17.7 Ω × cm² for co-treatment) compared to untreated control, and decreased BBB permeability, mediated by increased claudin-5 expression. In summary, this humanized triculture BBB model, enhanced by GDNF and cAMP, offers an alternative for exploring <i>in vitro</i> drug penetration into the human brain.