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

Abstract

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