Isolation of Cerebral Capillaries from Human Brain: An Optimized Method to Extract Cerebral Capillaries from Human Brain Tissue

To begin this procedure document the weight of the human brain tissue, which should be about 10 grams. Under the microscope, carefully remove all the meninges with forceps, and use a scalpel to cut off the white matter. Then, carefully cut up the brain tissue and mince it with a scalpel for about 5 minutes.

Transfer the brain tissue to the tissue grinder and add 30 milliliters of isolation buffer. Subsequently, homogenize each sample with 100 strokes at 50 rpm. Do not stir in air to prevent bubbles. Document the time every 25 strokes and the total time needed for 100 strokes. Afterward, transfer the homogenate to a Dounce homogenizer on ice. Homogenize the suspension for about 20 strokes and avoid bubbles.

Then, distribute the brain homogenate equally into four 50-milliliter centrifugation tubes and document the total volume of the homogenate. Ensure to add the correct volume of Ficoll. Too little or too much Ficoll will result in an incorrect density to separate capillaries from cellular debris and thus will reduce the capillary pellet and limit the yield.

Use 10 milliliters of isolation buffer to rinse the pestle and homogenizer and distribute it into the four centrifugation tubes. Next, distribute 50 milliliters of density gradient buffer into the centrifugation tubes. Tightly close the centrifuge tubes with caps. Afterward, mix the homogenate, density gradient medium, and buffer by vigorously shaking the tubes. Centrifuge at 5,800 x g for 15 minutes at 4 degrees Celsius. Subsequently, discard the supernatant and resuspend each pellet in 2 milliliters of 1% BSA.

After resuspending the pellet, filter the suspension through the 300-micrometer mesh. Capillaries are filtered through the mesh, whereas larger vessels and larger brain debris remain on the mesh. Carefully wash the mesh with up to 50 milliliters of 1% BSA, then discard the mesh. To separate the capillaries from red blood cells and other brain debris, distribute the capillary filtrate over 530-micrometer cell strain filters.

Capillaries are held back by this filter, whereas red blood cells, other single cells, and small brain debris pass through the filter and are collected in the filtrate. Next, wash each filter with 25 milliliters of 1% BSA. Pour all filtrates over the sixth filter to increase the yield. Wash each filter with 50 milliliters of 1% BSA. Keep the cell strain filters containing the capillaries and discard the filtrate. Then, turn the filters upside down and wash the capillaries with 50 milliliters of 1% BSA into 50-milliliter tubes.

Gently apply pressure with the pipette tip and move it across the filter to wash off the brain capillaries. Make sure to wash off all brain capillaries, especially from the rim of the filter. Avoid bubbles since this makes the filtration process more difficult and increases the chance of capillary loss. After collecting the capillaries, centrifuge all the samples at 1,500 x g for 3 minutes at 4 degrees Celsius. Remove the supernatant and resuspend the pellet in approximately 3 milliliters of isolation buffer.

Then, combine all resuspended pellets from one sample in a 15-milliliter conical tube and fill it with isolation buffer. Centrifuge again at 1,500 x g for 3 minutes at 4 degrees Celsius and wash two more times. Document the capillary purity with a microscope at 100X magnification and camera. The isolated brain capillaries can now be used for experiments, processed or be flash-frozen, and stored at minus 80 degrees Celsius in cryotubes for a minimum of 6 to 12 months.