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

Remove the brain from the skull of three 5-week-old Wistar rats and transfer the brains into a Petri dish filled with cold dissection buffer held on ice.

Dissect out the brains without the cerebellum and optic nerves. Then, cut each brain in half to separate the two cerebral hemispheres.

Under the stereomicroscope, make another cut to remove the midbrain and transfer all forebrains into a clean Petri dish on ice with dissection buffer.

Then, transfer one forebrain into a new Petri dish filled with cold dissection buffer. Hold it with the clamp and carefully detach the meninges from the edges of the forebrain.

Afterward, turn the forebrain over and carefully pull off the meninges without tearing them. Make sure that the forebrain is cleared of meninges and cut the remaining part of the optic nerves. The aim of this step is to remove the duvet of white matter to obtain a shell of cortex.

Start by clamping the forebrain at the edge and then carefully drag the forceps along the surface, taking care not to tear the surface of the cortex.

Then, make sure that the brain is free of the meninges by rolling it over to check that there are no residual meninges or big surface vessels.

Next, starting with the extraction of the three brains from their skull, the full time for the dissection should not take more than 2 hours for tissue preservation and cell survival. Transfer the cortices of three brains to 6 milliliters of cold dissection buffer in a 7-milliliter Dounce homogenizer.

Subsequently, dissociate the cortices with 10 up and down strokes with the first pestle of larger clearance, followed by 10 up and down strokes with the second pestle of smaller clearance.

Then, transfer 6 milliliters of the suspension into a new 50-milliliter Falcon tube and wash the Dounce homogenizer with 24 milliliters of cold dissection buffer. Divide the suspension into three Falcon tubes to obtain the equivalent of one cortex per tube.

Afterward, centrifuge at 1000 x g for 5 minutes at room temperature. Observation of the pellet shows a gradient of pink color, which represents the microvessel part of the cortices' homogenate.

Now, discard the supernatant and homogenize the pellet from one cortex with 1 milliliter of enzymatic solution containing a mix of collagenases, dispase, DNase type 1, and gentamicin.

Place the tubes in a shaker for 30 minutes at 37 degrees Celsius. Then, mix 1 milliliter of digest with 10 milliliters of 25% BSA/HBSS 1X and gently vortex the tubes. Subsequently, separate them by density-dependent centrifugation at 3,600 x g for 15 minutes at room temperature.

After the density-dependent centrifugation, the pellet contains the microvessels and the upper disk contains myelin, brain parenchyma, and residual microvessels.

Carefully transfer the upper disk and the supernatant into clean 50-milliliter Falcon tubes; take care not to aspirate the microvessel pellet.

Gently vortex the tube and repeat the density-dependent centrifugation. During this second centrifugation, keep the resulting pellet containing the microvessels at 4 degrees Celsius.

After the second density-dependent centrifugation, carefully discard the upper disk and the supernatant. Mix and resuspend the resulting pellets containing the microvessels from both centrifugations with 1 milliliter of cold HBSS 1X.

Next, wash the microvessels with 20 milliliters of cold HBSS 1X and centrifuge at 1,000 x g for 5 minutes. Then, discard the supernatant, taking care not to lose the microvessel pellet.

Homogenize the microvessel pellet from one cortex with 1 milliliter of the same enzymatic solution following a gentle vortex of the tubes. Further, digest the microvessels for 1 hour at 37 degrees Celsius in a shaker. After that, mix the digested microvessels from the three cortices into one tube.

Again, separate the mixture into two 50-milliliter Falcon tubes to obtain the microvessels extracted from the equivalent of one and a half cortices per tube. Then, centrifuge at 1,000 x g for 5 minutes at room temperature.

Just before plating the microvessels, take two coated T75 flasks from the incubator and aspirate the excess of coating. Discard the supernatant and resuspend the microvessel pellet in 1 milliliter of ECM.

Bring each tube to 10 milliliters with ECM supplemented with 4 micrograms per milliliter of puromycin to start the purification process and plate the microvessels in the flask.

Then, put the flasks in the incubator at 37 degrees Celsius and 5% carbon dioxide to allow microvessel adhesion to the collagen-fibronectin matrix.