Isolation and Culture of Primary Brain Pericytes: A Procedure to Isolate and Culture Pericytes from Bovine Brain Capillaries Without Endothelial Cell Contamination

Overview

In this video, we demonstrate the procedure to isolate and culture pericytes from bovine brain capillaries without endothelial cell contamination.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Seeding and Culturing of Bovine Capillaries

1. Day 0: Mix 0.7 mL of collagen IV stock with 6.3 mL of PBS. Add the solution to a T75-flask and leave the flask for 2 h at room temperature (RT) or leave it overnight at 4 °C.
2. Remove the collagen solution from the flask and wash three times with PBS.
3. Add 7 mL of fibronectin work solution and leave the flask for 30 min at RT. Then, remove the fibronectin solution and seed the capillaries immediately after.
4. During the 30 min waiting time, thaw one vial of capillaries in a 37 °C water bath.
5. When the capillaries are thawed, transfer immediately to a centrifugation tube with 30 mL of DMEM-comp and centrifuge for 5 min at 500 x g and RT. Remove DMEM-comp from the tube and resuspend the capillary pellet in 10 mL of fresh DMEM-comp.
6. Transfer the 10 mL suspension to the coated T75-flask and leave the capillaries to adhere to the bottom of the flask for 4–6 h in a 37 °C incubator at 10% CO₂.
   NOTE: The cell growth rate is higher at 10% CO₂ rather than the conventional 5% CO₂.
7. After 4–6 h of incubation inspect the flask under a light microscope. Fractions of capillaries should now be attached to the bottom of the flask (Figure 1, day 0).
8. Prepare GM and aspirate the DMEN-comp medium very careful from the capillaries and replace it with 10 mL of freshly made GM.
9. Day 2: Remove GM from the capillaries and replace with 10 mL of freshly made GM. Cellular outgrowth from the capillaries should be visible under a light microscope at this point (Figure 1, day 2–3).

2. Isolation of Primary Pericytes from Bovine Brain Capillaries

1. Day 4: Inspect the capillaries under a light microscope.        
   NOTE: The flask should now be approximately 60–70% confluent to provide an appropriate amount of pericytes (Figure 1, day 4). If this is not the case; replace the GM with 10 mL of fresh medium and leave the flask in the incubator for another day.
2. Aspirate the medium and wash the cells gently in PBS.
3. Add 2 mL of thawed Trypsin-EDTA for endothelial cells and leave the flask in the incubator for 1–3 min. Take out the flask frequently and observe with the microscope during this time period.  
   NOTE: The endothelial cells should round up and detach from the flask; pericytes should be visible as cells with a "ghost"-morphology and still be attached to the surface of the flask. This is a tricky and important step. It is essential to remove most endothelial cells to avoid contamination of the pericyte monoculture, but prolonged trypsinization can also detach the pericytes. The trypsinization time can vary slightly from time to time, and it is therefore of utmost importance to observe the flask frequently with the microscope during the treatment.
4. Gently tap the flask, when the endothelial cells have started to round up, to detach the loosened endothelial cells.
5. To stop the trypsinization, add 10 mL of DMEM-comp to the flask. Flush the flask carefully a few times with the medium to remove the endothelial cells. Aspirate the endothelial cell suspension from the flask. The endothelial cells can now be used for other purposes.
6. Add 10 mL of DMEM-comp to the flask. Look under the light microscope to assure the pericytes are still present and attached to the bottom. Put the flask back into the incubator to allow the pericyte-enriched culture to grow.
   NOTE: It is important to observe the culture during the following days. If there is still a fair number of endothelial cells growing another trypsin-treatment can be performed.
7. Allow the pericyte monoculture to grow with change of DMEM-comp. medium every second day. Check the growth of the cells under the light microscope (Figure 1, day 5–8).

Representative Results

Figure 1: Culturing of capillaries and isolation of pericytes. Capillaries have been isolated from fresh bovine brain and seeded in culture flasks on day 0. Outgrowth from the bovine brain capillaries and the following isolation of pericytes were followed over days with a light microscope. Day 4a shows the endothelial cell growth prior to treatment with trypsin to remove endothelial cells and day 4b shows the remnants immediately after the treatment. Day 8a shows the focus plane, where any capillary remnants would be visible, whereas day 8b are focused on the plane where the growth of the pericytes is visible.

Materials

Name	Company	Catalog Number	Comments
PBS	Sigma Aldrich	D8537	Phosphate-buffered saline
Collagen IV	Sigma Aldrich	C5533
T-75 flask	Sigma Aldrich	CLS3972
Fetal bovine serum (FBS)	PAA/GE Healthcare	A15-101
Fibronectin	Sigma Aldrich	F1141
Trypsin-EDTA	Sigma Aldrich	T4299
Light microscope	Olympus	Olympus CK2	Upright light microscope with phase contrast
DMSO	Sigma Aldrich	471267
Heparin	Sigma Aldrich	H3149
Cell incubator	Thermo Fisher
Centrifuge	Thermo Fisher	Heraeus Multifuge 3SR+	Standard large volume centrifuge for spinning down cells
Dulbecco's Modified Eagles Medium	Sigma Aldrich	D0819
Penicillin G Sodium/Streptomycin sulfate	Sigma Aldrich	P0781
MEM nonessential amino acids	Sigma Aldrich	M7145