Endothelial Cell Culture from Rat Brain Microvessels: A Procedure to Isolate and Culture Endothelial Cells from Microvessels of Rat Brain

To isolate brain endothelial cells, first, transfer a harvested rat brain into a dish containing chilled buffer.

After removing the cerebellum and optic nerves, divide the brain into cerebral hemispheres. Then, separate the midbrain from the forebrain.

Remove the meninges and the myelin[-containing white matter from the forebrain, leaving behind the brain cortex for isolating the microvessels.

Next, homogenize the cortex to break the nervous tissue and constituents.

Centrifuge the homogenate to remove cellular debris and myelin.

Subsequently, treat the tissue pellet with a digestion cocktail containing collagenase, dispase, and deoxyribonuclease enzymes. 

Collagenases and dispases digest the extracellular matrix that embeds the microvessels, loosening the microvessel fragments from the tissue. Deoxyribonucleases degrade any free DNA released from lysed cells.

Mix the desired volume of the digested homogenate with an appropriate density gradient medium and centrifuge.

Discard the myelin and brain parenchymal cell-containing uppermost layer, followed by the supernatant.

Now, treat the microvessel-containing pellet with the digestion cocktail to completely release the microvessel fragments comprising endothelial cells and pericytes. 

Centrifuge the suspension. Resuspend the microvessel fragment-containing pellet in a suitable media supplemented with puromycin - an antibiotic.

Seed the suspension into an extracellular matrix protein-coated plate and incubate.

The microvessel fragments adhere to the coating in the plate. Puromycin selectively eliminates pericytes, while the media components facilitate the proliferation of endothelial cells.