Isolation and Culture of Primary Brain Pericytes: A Procedure to Isolate and Culture Pericytes from Bovine Brain Capillaries Without Endothelial Cell Contamination
Transcrição
In the central nervous system, pericytes are multi-functional cells that envelop the endothelial cells, which form the inner cellular lining of the capillaries. Pericytes help regulate blood flow and maintain brain homeostasis.
To isolate pericytes, begin by taking frozen bovine brain capillaries in a vial. After thawing, transfer the contents to a tube containing the desired media. Centrifuge to pellet the capillaries. Remove the freezing media-containing supernatant. Resuspend the capillary pellet in media and seed the suspension into an extracellular matrix protein-coated flask.
Incubate to allow the capillaries to adhere to the coating in the flask. Replace the media with heparin-containing media. In culture, the extracellular matrix protein coating promotes pericytes to migrate away from the capillary fragments and proliferate, while heparin stimulates endothelial cell migration.
Replace the spent media with heparin-containing media. Over time, pericytes project elongated processes that adhere strongly to the flask. With limited substrate space, endothelial cells proliferate and form a contact-inhibited monolayer. Upon achieving appropriate pericyte density, remove media and wash the flask with buffer.
Use trypsin-EDTA to detach the loosely-bound endothelial cells from the flask. Subsequently, add media to neutralize trypsin-EDTA activity. Wash with media repeatedly to remove the detached endothelial cells. Add media and ensure that pericytes remain attached to the flask. Incubate the pericyte-enriched culture to proliferate.
