Isolating and Culturing Mouse Cerebral Pericytes

Samenvatting

Source: Mehra, A., et al., A High Output Method to Isolate Cerebral Pericytes from Mouse. J. Vis. Exp. (2020)

This video demonstrates a method for isolating cerebral pericytes from a mouse brain. The isolated cells are cultured in pericyte-specific media to promote pericyte proliferation.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Preparation of solutions

1. Prepare 500 mL of Washing Buffer A (WBA): 10 mM HEPES solution in Hank's Balanced Salt Solution (HBSS). Store at 4 °C.
2. Prepare 500 mL of Washing Buffer B (WBB): 0.1% Bovine Serum Albumin (BSA) with 10 mM HEPES solution in HBSS. Store at 4 °C.
3. Prepare 300 mL of 30% dextran solution in WBA by mixing the solution overnight at room temperature. Autoclave the solution at 110 °C for 30 min before use. After autoclaving, let the solution rest at room temperature for 2-3 h. Store the solution at 4 °C.
4. Prepare 100 mL of 0.1% BSA solution in cold dextran solution. Shake vigorously for 3-4 min and store at 4 °C.
5. Prepare complete Dulbecco's Modified Eagle Medium (DMEM) culture media by dissolving 20% calf serum, 2 mM glutamine, 50 µg/mL gentamycin, 1% vitamins, 2% amino acids Basal Medium Eagle (BME) in Basal DMEM media and store at 4 °C. Add 1 ng/mL Basic fibroblast growth factor (bFGF) prior to use.
6. Prepare complete pericyte media by adding pericyte growth supplements (provided with the pericyte media) and 20% Fetal Calf Serum (FCS) in pericyte culture basal media and store at 4 °C.

2. Brain tissue recovery and removal of meninges

1. For consistency, use mice of similar age and same gender in every batch of extraction. Use a pathogen-free animal shelter and provide ad libitum access to water. To ensure efficiency and minimal use of animals, avoid loss of tissue material.
2. Euthanize C57BL/6J, 4-6 weeks old, male mice (Janvier labs, Le Genest-Saint-Isle, France).
3. Quickly excise the brain tissue in sterile conditions, avoid any damage to the tissue. Carefully place the tissue in 40 mL of cold phosphate buffered saline (PBS).
4. Transfer the brain tissue in cold PBS to a Petri dish (100 mm x 15 mm).
5. Place the brain tissue on a sterile dry lint-free wipe and with curved tip forceps, remove the cerebellum, striatum and occipital nerves.
   1. Remove all the visible meninges with a cotton swab. Place the brain tissue upside down and open the lobes with a cotton swab using outward light strokes. Remove all the visible blood vessels.
   2. Place the meninges free brain tissue in a Petri dish (100 mm x 15 mm) with 15 mL of cold WBB.

3. Homogenization

1. Transfer the tissue to a Dounce tissue grinder mortar tube and add 3-4 mL of WBB with forceps.
2. Mince the tissue with a 'loose' pestle 55 times. Rinse the 'loose' pestle with WBB. Now mince the slurry with a "tight" pestle 25 times.
3. Divide the slurry equally into two 50 mL tubes and add 1.5x volume of cold 30% BSA-dextran, vigorously shaking the tubes to mix the slurry.

4. Isolation of the vascular fraction

1. After vigorously shaking the tubes, centrifuge the tubes for 25 min at 3,000 x g and 4 °C.
2. Transfer the supernatant (along with the top myelin layer) to 2 new tubes, and centrifuge the tubes for 25 min at 3,000 x g and 4 °C. Preserve the pellets from the first centrifugation by adding 3 mL of cold WBB (keep the pellet at 4 °C).
3. Repeat step 4.2 and carefully preserve the pellets from 2nd centrifugation.
4. Discard the dextran and the myelin with tissue debris from the tubes from step 4.3. Preserve the pellets in cold WBB.
5. Pool the contents of tube 1 and tube 2 from step 4.1 and make up to final volume of 10 mL with cold WBB.
6. Repeat this step for tubes from step 4.2 and step 4.3.
   NOTE: Finally, there are 3 tubes from 3 centrifugations.
7. Dissociate the pellet using 6 up-and-down strokes using a 10 mL pipette until no visible clumps of pellets remain.
8. Filter the cell suspensions of each tube using a vacuum filter assembly and a nylon mesh filter.
   NOTE: This filtration step is important in order to remove longer/larger vessels via the mesh filter.
9. Recover and resuspend the capillaries by scraping the filter with the help of a flat tip forceps or a scraper in WBB at room temperature. Filter the suspension with a fresh filter to recover more capillaries
10. Divide the filtrate equally into two tubes and centrifuge for 7 min at 1,000 x g and RT.
    NOTE: During this centrifugation step, prepare the enzymatic solution. Determine the volume of WBB required in accordance with the number of animals used (see Table of Materials). Add 1x of DNase 1 and 1x of Tosyl-L-lysyl-chloromethane hydrochloride (TLCK) (see Table of Materials) in WBB and pre-warm to 37 °C.
11. Collect the pellets from step 4.10 in one tube with pre-warmed WBB with enzymes. Add pre-warmed 1x collagenase/dispase. Place the tube in the shaking table water bath at 37 °C for precisely 33 min.
12. Stop the enzyme reaction by adding 30 mL of cold WBB. Centrifuge the suspension for 7 min at 1,000 x g and RT.
13. Discard the supernatant carefully and dissociate the pellet in WBB with 6 up and down strokes using a 10 mL pipette. This step should be less rigorous and comparatively faster.
14. Centrifuge the suspension for 7 min at 1,000 x g and RT.
    NOTE: During this step, discard the coating from the culture dishes and rinse them once with DMEM at room temperature. Coat cell culture dishes for at least 1 h at RT.
15. Discard the supernatant from the tube obtained at step 4.14, and dissociate the pellet in new complete DMEM media, plate the cells (Day 0, P0) in 9 wells of 6-well plates (1 well of 9.6 cm2 each).

5. Proliferation of cerebral pericytes

1. Maintain the cell cultures at 37 °C and 5% CO₂ in a sterile incubator. Replace the culture media after 24 h (day 1) of plating the cells by carefully removing the debris. After day 1, change the culture media in every 48 h.
2. Observe the cell culture for at least 7-8 days. By this time, cellular growths on the top of endothelial unilayer should be observable.
3. Passage the cells on day 8-10 (depending on the confluency) in pericyte culture medium to passage 1 (P1) on gelatin-coated culture plates. Change the culture media in every 2 days. Observe the cells for 6-7 days. Cells are consecutively split again to passage 2 (P2) on day 17 [and passage 3 (P3) on day 24 only if required], grown in pericyte medium in gelatin coated plates.
   NOTE: Cells shall be ready for experiments/observation at nearly 80-90% confluency.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Amino acids BME	Sigma	B-6766	Store at 4 °C.
Basal DMEM media	Invitrogen	316000083	Store at 4 °C.
Basic fibroblast growth factor	Sigma	F-0291	Store at -20 °C.
BSA	Sigma	A-8412	Store at 4 °C.
Collagenase dispase	Sigma	10269638001	Prepare a 10x stock solution in sterile PBS-CMF. Filter the solution with a 0.22 μm syringe filter and store at -20 °C. Note: For the enzyme digestion step of the protocol, for every set of 10 mice for extraction, 300 µL of 10x collagenase dispase is required.
Dextran	Sigma	31398
DNase I	Sigma	11284932001	Prepare a 1000X stock solution by dissolving 100 mg in 10 ml sterile water and store at -20°C.
Gelatin	Sigma	G-2500	Prepare the working coating by making a 0.2% gelatin solution in sterile PBS-CMF (8 g/L NaCl, 0.2 g/L KCl, 0.2 g/L KH2PO4, 2.86 g/L NaHPO4 (12 H2O), pH 7.4). Autoclave the solution for minimum 20 minutes at 120 °C and store at room temperature. Culture dishes to be coated for at least 4 hours at 4 °C.
Gentamycin	Biochrom AG	A-2712	Store at 4 °C.
Glutamine	Merck	I.00289	Store at -20 °C.
HBSS	Sigma	H-8264	Store at 4 °C.
HEPES	Sigma	H-0887	Store at 4 °C.
Matrigel	BD Biocoat	354230	Prepare a working coating solution of Matrigel by diluting stock in cold DMEM at 1:48 ratio with its final concentration to be 85 µg/cm2. Cell culture dishes should be coated at least for 1 hour at room temperature.
Pericyte Medium-mouse	Sciencell research laboratories	1231	Store at 4 °C.
Tosyl Lysin Chloromethyl Ketone	Sigma	T-7254	Prepare a 1000X stock solution in WBA by dissolving 16 mg in 10.88 mL of WBA to make a 4 mM solution and store at 4 °C.
Vitamins	Sigma	B-6891	Store at -20 °C.
Filtration tools	Sefar, Nylon mesh, 60-micron porosity
Laboratory equipment	Swing bucket rotor centrifuge
Water bath with agitator
Laminar Flow Hood : BSL2
Glassware (all components to be heat sterilized)	Dounce Tissue Grinder With Glass Pestle
Pestle I: 0.0035 - 0.0065 inches
Pestle II: 0.0010 - 0.0030 inches
Vacuum filter assembly with coarse porosity fritted glass filter support base