Blood-Brain Barrier Mimic Permeability Assay: An In Vitro Assay to Assess the Permeability of BBB Mimic to Tracer Molecules

The blood-brain barrier, or BBB, consisting of endothelial cells, pericytes, and astrocytes, regulates the influx and efflux of biological molecules and helps maintain brain homeostasis.

To assess the permeability of the BBB model in vitro, begin with an in vitro BBB model consisting of adhered endothelial cells on the upper or blood side of the porous membrane insert and adhered astrocytes on the basal or brain side of the membrane. 

The endothelial cells with their extracellular matrix contact the astrocytes, forming the BBB mimic.  

Transfer the desired volume of media from the blood and the brain side into a black multi-well plate for optimal fluorescence measurement.

Replace the media from the blood side of the culture with media containing small molecular weight fluorescent tracer molecules.

In culture, the endothelial-astrocyte cell contacts enhance the cellular barrier and stabilize the tight barrier, limiting the diffusion of tracer molecules to the brain side of the culture.

Further, collect suitable volumes of media from the blood and the brain side at the desired time points. Replace both sides with media to maintain media volume.

Finally, use a multi-well plate reader to measure the fluorescence of the collected media to evaluate the permeability of the BBB model.

The lower fluorescence intensity from the brain side suggests lower permeability of the cellular barrier.