Development of an in-vitro High-Throughput Screening Assay for the Identification of Modulators of the Blood-Brain Barrier Endothelium Integrity

The blood-brain barrier (BBB) controls the content of brain interstitial fluid due to the presence of high-resistance tight junction proteins between the endothelial cells of brain capillaries. Severalmodels using different cell-lines were developed to study the BBB biology, these models werecomplex and their use for high-throughput screening (HTS) has proven to be challenging. Therefore, we developed an in-vitro BBB model that is practical and reliable for HTS. The mouse brainendothelial cells (bEnd3) grown on 96-well plate inserts were upgraded and optimized for HTSassay. Using Lucifer Yellow (LY) permeation assay, 3 different compounds libraries that include3000 compounds were screened for hits that have the potential to modulate the BBB model integrity. To evaluate the model ability to identify compounds that increase LY permeation, mannitol wasused as a positive control for disruptors. The model performance in this assay was high with Z'factor above 0.5 and high S/N and S/B ratios. Alternatively, hydrocortisone was used as a positivecontrol for compounds that enhance the barrier function as it is known to improve endotheliumtightness. The Z' factor determined with hydrocortisone was 0.3 with lower S/N and S/B ratioscompared to mannitol. The primary screen has identified several hundred of modulators. Thesecondary screen could identify 13 compounds as potent enhancers of the monolayer integrity withEC50 values less than 10 μM. In conclusion, an HTS-BBB model was developed and used forcompounds screening to identify compelling hits for further evaluation for their effect on the BBB.