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

Abstract

Source: Hørlyck, S. et al. Culture of Brain Capillary Pericytes for Cytosolic Calcium Measurements and Calcium Imaging Studies. J. Vis. Exp. (2020)

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 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. Remove the collagen solution from the flask and wash three times …

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 an…

Materials

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