Isolation and Culture of Primary Brain Pericytes: A Procedure to Isolate and Culture Pericytes from Bovine Brain Capillaries Without Endothelial Cell Contamination
Transcription
Begin by thawing one vial of capillaries in a 37 degrees Celsius water bath. Once the capillaries have thawed, transfer them to a centrifuge tube with 30 milliliters of DMEM-comp and centrifuge them for 5 minutes at 500 x g. Then, remove the medium from the tube and resuspend the pellet in 10 milliliters of fresh DMEM-comp. Transfer the suspension to a coated T75-flask and allow the capillaries to adhere to the bottom for 4 to 6 hours in a 37 degrees Celsius incubator supplied with 10% carbon dioxide. After the incubation, inspect the flask under a light microscope.
Fractions of capillaries should now be attached to the bottom of the flask. On day 4, after seeding the capillaries, inspect them under the microscope to ensure that they are approximately 60% to 70% confluent. Aspirate the medium and gently wash the cells with PBS. Add 2 milliliters of thawed Trypsin-EDTA and leave the flask in the incubator for 1 to 3 minutes, taking the flask out frequently and observing cell detachment with the microscope. When the endothelial cells start to round up, gently tap the flask to detach them.
When the cells have detached, stop trypsinization by adding 10 milliliters of DMEM-comp to the flask. Flush the flask a few times with the medium and aspirate the endothelial cell suspension, which can now be used for other purposes. Add 10 milliliters of DMEM-comp to the flask and check it under the light microscope to assure that the pericytes are still present and attached to the bottom. Then, put the flask back into the incubator to allow the pericyte-enriched culture to grow.
