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.

First, collect 100 microliters of the medium from both the blood and the brain sides of the BBTB mimic and transfer each to a separate flat-bottomed, black 96-well plate for subsequent fluorescence measurements.

Next, prepare a 50-micromolar solution of sodium fluorescein in EBM minus, making sure to prepare 2.5 milliliters per well. Prewarm the solution to 37 degrees Celsius and replace the media from the blood side of the inserts with media containing this sodium fluorescein solution. Start a timer as soon as the medium is replaced.

Carefully collect 100 microliters of media from both the blood and brain sides of the inserts at 5 minutes, 30 minutes, 60 minutes, and 120 minutes. Transfer each sample to separate wells of the appropriate black 96-well plate. Use a plate reader to quantify the fluorescence from the collected samples.