An Improved In Vitro Blood-Brain Barrier Model for the Evaluation of Drug Permeability Using Transwell with Shear Stress

TL;DR

This study improves an in vitro model of the blood-brain barrier by adding shear stress, enhancing its ability to predict drug permeability for CNS-targeting drugs.

Contribution

A novel dish design for an orbital shaker is introduced to apply shear stress and improve transwell BBB models.

Findings

The annular shaking-dish model significantly improved cell-layer integrity compared to traditional transwell models.

The model showed robust permeability evaluation of 14 compounds, aligning with published in vivo data.

Efflux transporter activity and junctional protein expression were maintained in the optimized model.

Abstract

The development of drugs targeting the central nervous system (CNS) is challenging because of the presence of the Blood-Brain barrier (BBB). Developing physiologically relevant in vitro BBB models for evaluating drug permeability and predicting the activity of drug candidates is crucial. The transwell model is one of the most widely used in vitro BBB models. However, this model has limitations in mimicking in vivo conditions, particularly in the absence of shear stress. This study aimed to overcome the limitations of the transwell model using immortalized human endothelial cells (hCMEC/D3) by developing a novel dish design for an orbital shaker, providing shear stress. During optimization, we assessed cell layer integrity using trans-endothelial electrical resistance measurements and the % diffusion of lucifer yellow. The efflux transporter activity and mRNA expression of junctional proteins (claudin-5, occludin, and VE-cadherin) in the newly optimized model were verified. Additionally, the permeability of 14 compounds was evaluated and compared with published in vivo data. The cell-layer integrity was substantially increased using the newly designed annular shaking-dish model. The results demonstrate that our model provided robust conditions for evaluating the permeability of CNS drug candidates, potentially improving the reliability of in vitro BBB models in drug development.

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