Aortic Ring Assay

One day before the experiment:

1. Allow BME (reduced growth factor, Cultrex, Trevigen) to thaw on ice or at 4°C; once thawed keep on ice to prevent re-solidifying.

Day of the experiment:

1. Mice (6-7 weeks old) are to be anesthetized and bled out.
2. Remove thoracic aorta into a Petri dish filled with cold sterile PBS.
3. Perform mechanical cleaning from surrounding fat tissue. Note, avoid drying of Aorta at any time.
4. Using a surgical blade, slice Aorta evenly into 1 mm rings. The cut must be uniform and clean.
5. Transfer rings to fresh cold PBS and keep on ice at all times.
6. Using pre-cooled pipette tips, add to each well in a 48-well plate a rounded drop of 150 ml cold BME. Allow the BME drop to solidify at 37°C for 20-30 minutes.
7. Place a single aortic ring in the top center of each dome. Incubate for 10 minutes at 37°C.
8. On top of each ring add an additional 150 ml BME. Incubate for 20-30 minutes at 37°C.
9. Prepare medium: Human endothelial serum free medium (GIBCO, Carlsbad, CA) supplemented with 2% FCS, 50 units/ml penicillin and 50 μg/ml streptomycin (Cellgro).
10. To each well add 500 ml medium containing either:
    1. Experimental samples.
    2. Positive control: Endothelial cell growth supplement (ECGS, final concentration 200 μg/ml, BD Biosciences).
    3. Negative control: Medium alone.
11. Incubate plate at 37°C for 12 days, photograph rings between days 6 and 12 under standard phase contrast stereoscope.
12. Optional: Collect supernatants.

Notes:

1. Keep sterile conditions at all times.
2. Avoid introduction of bubbles into the BME.

Aortic ring assay is a beneficial tool on the way to evaluate angiogenic as well as anti-angiogenic factors. The vessels that grow out from aortic rings recruit smooth muscle cells and pericytes to associate with the endothelial cell tube, meaning that they are anatomically similar to neovessels in vivo. Furthermore, the mouse aortic ring assay holds the advantage of the vast array of transgenic tools available for this species. Yet, there are several disadvantages to the aortic ring assay. First, vessels outgrowth in vivo occurs from microvessels and not from major vessels such as the aorta. Second, inconsistency in the handling of the rings and the amount of surrounding tissue remaining on the vessel can influence vessel outgrowth. Rings from different aortas or different mice's ages and strains can also interfere with the angiogenic responses. And lastly, Angiogenic vessel outgrowth occurs in three dimensions, which makes it non-ideal to photograph and quantify. Taken together, when performed with sufficient internal controls and with multiple repeats, this assay is relatively simple, non-expensive, highly informative and significantly superior to endothelial cultures in its biological complexity and relevance.