Design of experiment based optimization of an in vitro direct contact triculture blood brain barrier model for permeability screening

The in vivo restrictive properties of the blood brain barrier (BBB) largely arise from astrocyte and pericyte synergistic cell signaling interactions that underlie microvessel endothelial cells (BMEC). In relevant direct contact between astrocytes, pericytes, BMECS, to our knowledge, has not been established conventional Transwell® based vitro screening models BBB. We hypothesize a design experiments (DOE) optimized layered triculture model will offer more relevance for comparison indirect models. Plating conditions including seeding density all three types, matrix protein, culture time were assessed utilizing DOE approach. A second set methods influence medium additives on properties. was further p-glycoprotein function using substrate inhibitor along with BBB paracellular transcellular markers at varying permeation rates. optimization revealed length post plating correlated highest tightness. addition, layer influenced tightness earlier times culture, its impact decreased as is extended. At optimal conditions, P-gp ability differentiate positive negative permeants. have demonstrated implementation biologically systems an expedited method establish multi-component reveals physiologically layering types practical compared incorporate physical barriers types. Additionally, permeants suggests this may be enhanced tool potential neuroactive compounds.