This method can help answer key questions in the field of blood-brain barrier research, such as blood-brain barrier dysfunction, barrier leakage and signaling pathways that are specific for the human blood-brain barrier. The main advantage of this technique is that it allows the study of intact, physiologically active human brain capillaries that have been isolated from fresh 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 five 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 no 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 palette 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 times gravity for 15 minutes at four degrees Celsius. Subsequently, discard the supernatant and resuspend each pellet in two 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 five 30 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 times gravity for three minutes at four degrees Celsius.
Remove the supernatant and resuspend the pellet in approximately three 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 times gravity for three minutes at four degrees Celsius and wash two more times.
Document the capillary purity with a microscope at 100 times 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 six to 12 months. This figure shows a representative transmitted light image of a human brain capillary with an attached pericyte and a red blood cell in the lumen.
The isolated human brain capillary was immunostained for PGP using C219 as the primary antibody and the nuclei were counterstained with DAPI. While attempting this procedure, it's important to remember to avoid loss of capillaries during pipetting and filtration steps to ensure a good capillary yield. Following this procedure, isolated human brain capillaries can be utilized for a wide range of studies such as pharmacological and physiological studies, as well as genomic, proteomic and cellomic studies.
This technique paved the way for researchers in the blood-brain barrier field to study barrier function in human brain capillaries as close to the human in vivo situation as possible. Don't forget that working with human brain tissue poses the risk of infection from blood-borne pathogens. Safety precautions should be taken, such as wearing a lab coat, face shield and gloves, as well as handling tissue samples in a BSL-2 setting while performing this procedure.
