Recently, the role of cerebral pericytes has become evident in neurological disorders and brain function. Therefore, the development of a reliable method of culture for these cells is imperative. Our protocol for the extraction of murine cerebral pericytes represents a reliable tool for in vitro studies and for providing pericytes with high purity and cell yield.
Demonstrating the procedure with Anupriya Mehra will be Lucie Dehouck, a technician from my laboratory. Begin by placing the brain from a specific pathogen-free four to six-week-old male C57BL/6 mouse onto a sterile dry lint-free wipe and using curved tip forceps to remove the cerebellum, striatum and occipital nerves. Use a cotton swab to remove all of the visible meninges and turn the brain tissue upside down.
Open the lobes with light outward strokes and remove all of the visible blood vessels. Then place the meninges-free brain tissue in a 100 millimeter Petri dish containing 15 milliliters of cold washing buffer B.To homogenize the tissue, transfer the brain sample into a Dounce tissue grinder mortar tube and add three to four milliliters of washing buffer B to the tube. Use a loose pestle to mince the tissue 55 times, then rinse the pestle with washing buffer B and mince the tissue slurry with a tight pestle 25 times.
At the end of the homogenization, equally aliquot the slurry between two 50 milliliter tubes and add 1.5 times the volume of cold 30%bovine serum albumin dextran. Then vigorously shake the tubes to mix the slurry. To isolate the vascular fraction, sediment the cells by centrifugation and transfer the supernatants into two new tubes.
Store the pellet in washing buffer B on ice. Resuspend the pellets in three milliliters of cold washing buffer B on ice. Centrifuge the harvested supernatants and repeat this step further one more time.
Discard the supernatants and resuspend the pellets in three milliliters of washing buffer B.Then pool the resuspended pellets and bring the final volume of the pooled cell suspension to 10 milliliters with fresh cold washing buffer B.Further dissociate the cells with a 10 millimeter pipette six times until no remaining clumps of pellets are visible and use a vacuum filter assembly and a nylon mesh strainer to filter the cell suspension. Place the strainer in a Petri dish and clear the mesh with fresh washing buffer B and scraping to recover any capillaries. Then perform a second filtration with a fresh filter.
Divide the filtrate equally between two tubes and collect the cells by centrifugation. After discarding the supernatants, pool the pellets into a single tube containing prewarmed washing buffer B with enzymes and add prewarmed Collagenase/Dispase to the tube. Place the tube in a shaking table water bath for precisely 33 minutes at 37 degrees Celsius before stopping the reaction with 30 milliliters of cold washing buffer B.Collect the cells by centrifugation and carefully discard the supernatant.
Quickly but carefully resuspend the pellet in washing buffer B six times and centrifuge the suspension again. After discarding the supernatant, resuspend the pellet in 18 milliliters of complete DMEM medium and seed two milliliters of cell suspension in complete DMEM in nine wells of three Matrigel coated six-well plates. Place the cell cultures in a sterile 37 degree Celsius 5%carbon dioxide incubator for 24 hours before carefully removing the debris from each well and add fresh DMEM media.
After 48 hours, change the medium in each well every 48 hours. On day eight to 10 when the cells reach 100%confluency, passage the cells in pericyte culture medium onto new gelatin coated six-well plates and return the cells to the cell culture incubator for an additional six to seven days with monitoring. Then split the cells again on day 17 in pericyte medium into new gelatin coated plates.
From passage zero to passage two, there are specific morphological characteristics by which the endothelial cells and the subsequent gradual increase in pericytes can be identified. In a passage zero culture, elongated endothelial cells developing from microvessels are in abundance. This abundance is reduced after passage one and absent after passage two.
On the contrary, pericytes appear as rare quadrilateral cells in early cultures and become abundant by passage two. Evaluation of the expression of NG2, CD146, and PDGFR beta can be used to assess the purity of the pericyte culture by quantitative PCR, immunocytochemistry, and Western blot. The enzyme digestion is a critical step for the success of protocol.
Be sure to strictly monitor the enzyme concentrations and incubation time.
