An In Vitro Model for Studying Pathogen Crossing of the Blood-Brain Barrier and Brain Cell Infection

Take an in vitro blood-brain barrier, or BBB, model comprising two compartments separated by a porous membrane.

The upper or luminal compartment contains microvascular endothelial cells. These cells express tight junctions, mimicking the blood-brain barrier endothelium that limits the passage of cells and molecules.          

The lower or abluminal compartment contains neurons, astrocytes, and microglia, representing the brain parenchyma.

Introduce a neurotropic virus into the luminal compartment to simulate a blood-borne infection and incubate.

The virus is endocytosed by the endothelial cells, undergoes intracellular trafficking within vesicles, and is released on the abluminal side.

Post-incubation, remove the upper chamber. Sample the medium from the abluminal side to assess the viral quantity, confirming the successful crossing of the endothelial layer.

Add fresh medium and incubate again.           

The virus enters the neural cells via endocytosis, replicates, and releases viral progeny.

Quantify the increased extracellular viral load to confirm viral neuroinvasion and replication.

After setting up a BBB mini brain device, as demonstrated, add 3,500 plaque forming units of the French neurotropic virus strain of yellow fever virus, diluted in 50 microliters of 2% FBS endothelial cell medium very carefully on top of the luminal compartment. Inoculate the control BBB mini brain with 50 microliters of 2% FBS endothelial cell medium without virus. Then, place the virus exposed BBB mini brain device in the incubator for 24 hours.

The next day, remove the inserts to determine the endothelial cell permeability in a companion well, as just demonstrated, and save 1 milliliter of sample from each well to determine the virus titer in the abluminal compartment.

Under the insert gently to avoid leakage of the luminal compartment into the abluminal compartment.