Isolation and culture of rat coronary microvascular endothelial cells (CMVE) 

 CMVE were isolated from Wistar rat hearts by digestion of primary cell isolation kit.
1. Hearts mounted on a Langendorff apparatus were perfused at 37°C with a solution of the following composition (in mM): NaCl 118, KCl 4.7, NaH^₂PO4 1.2, MgSO^₄ 1.2, NaHCO^₃ 25, glucose 11, pH 7.4 (gassed with 95% O2/5% CO^₂)-Buffer 1.
2. Epicardial mesothelial cells were devitalised with 70% (v/v) ethanol.
3. After flushing out blood from the coronary circulation, perfusion was changed to Buffer 1 with added CaCl^₂ 0.25 μM and collagenase 0.04%, which was recirculated for 30 min.
4. Ventricles — excluding any visible large vessels — were then chopped into 15 ml of recirculating solution containing BSA (200 mg), and triturated gently during a 10 min incubation period at 37°C.
5. The suspension was filtered through nylon gauze and centrifuged (150 g, 3 min) to sediment myocytes.
6. The supernatant, including added BSA (100 mg), trypsin 0.01%, and CaCl^₂ 50 μM, was incubated at 37°C for 15 min with stirring.
7. The CMEC pellet was obtained by centrifugation (1000 g, 10 min), washed twice in Buffer 1 with CaCl^₂ 250 μM and 500 μM respectively, and resuspended in 40 ml Primary cell medium with 10% FBS, benzylpenicillin 250 U/ml, streptomycin 250 μg/ml, amphotericin B 12.5 μg/ml, and gentamycin 50 μg/ml.
8. Cell suspensions were plated in 25 or 75 cm2 tissue culture flasks and incubated at 37°C.
9. After 1 h, unattached cells and debris were washed off with 0.9% saline. Cultured cells formed confluent monolayers with a ‘cobblestone’ morphology within 5–7 days.
10. Cells were then trypsinised and subcultured to confluence in fresh flasks.
11. Cultured CMVE were characterised as endothelial by their typical ‘cobblestone’ morphology, and by the uptake of fluorescently labeled acetylated low-density lipoprotein by >99% of cells.
12. CMVE stained negatively for smooth muscle α-actin, and rapidly formed capillary-like tubes on the basement membrane preparation Matrigel.

References
1. Piper HM, Spahr R, Mertens S, Krutzfeldt A, Watanabe H. Cell Culture Techniques in Heart and Vessel Research. In: Piper HM, editor, Berlin; Springer-Verlag, 1990, 158-177.
2. Ulvi Bayraktutan, Nick Draper, Derek Lang and Ajay M. Shah. Expression of a functional neutrophil-type NADPH oxidase in cultured rat coronary microvascular endothelial cells. Cardiovasc Res, 1998; 38: 256-262.