Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies

Our research deals with angiogenesis, the process of formation of new vessels from preexisting vessels. It plays a critical role in many physiological and pathological processes, such as abnormal development, atherosclerosis, tumor development, et cetera. The construction of a reliable model to evaluate the extent of angiogenesis is critical for medical research or the discovery of medicines for angiogenesis-dependent diseases.

However, most of the existing models are complex, expensive, and require specialized equipment. Here we present a modified matrix gel plug assay that can be used to investigate angiogenesis and vessel permeability without staining, in a single, more intuitive, as well as efficient manner. Begin by preparing the complete endothelial culture medium, using 460 milliliters of endothelial cell medium, and adding into it 50 milliliters of FBS, five milliliters of penicillin streptomycin, and five milliliters of endothelial cell growth supplement.

The prepared media can be stored at four degrees Celsius for a month. Next, in a 100 millimeter tissue culture dish containing 0.1 million vascular cells add eight milliliters of the prepared complete endothelial culture medium. And culture the cells at 37 degrees Celsius and 5%carbon dioxide until 70%confluency is achieved.

Then, remove the culture medium from the dish and rinse the cells twice with PBS to eliminate unattached cells and debris. After removing the PBS, add three milliliters of 0.25%trypsin containing 2.21 millimolar EDTA to the cells, and incubate the dish at 37 degrees Celsius for one minute. Verify cell detachment under a light microscope at 40 times magnification.

Neutralize the trypsin with seven milliliters of complete endothelial culture medium, and gently rinse the cells off the culture dish. Centrifuge the cell suspension after collecting it in a 15 milliliter tube at 400G for 10 minutes. Resuspend the cells in five milliliters of complete endothelial culture medium after removing the supernatant.

Then, count the cells using a hemocytometer, and transfer a calculated volume of suspension containing 2 million cells to a sterile 1.5 milliliter tube. Pellet the cells by centrifuging the suspension at 400G for five minutes before removing the supernatant. The vascular cells are now ready for mixing with the matrix gel for matrix gel plug assay.

To begin, prepare 10 times M199 culture medium by dissolving M199 powder to 10 times concentration in 90 milliliters of deionized water, followed by the addition of 10 milliliters of FBS. Filter the solution through a 0.22 micron filter. The filtered medium can be stored at four degrees Celsius for up to two months.

For preparing the matrix gel with vascular cells, use about 2 million vascular cells. Cool the sterile 1.5 milliliter tube containing the appropriately prepared cell pellet in an ice bath. Thaw the matrix gel completely in an ice bath without mixing or vortexing it.

Similarly, cool the prepared 10 times M199 culture media along with the test reagent or drug of interest to four degrees Celsius. Next, maintaining a volume ratio of 8.8:1.0:0.2, combine the pre-cooled matrix gel with the pre-cooled 10 times M199 media containing 10%FBS and the pre-cooled test reagent to produce the matrix gel mixture. Finally, resuspend the pre-cooled cells in 400 microliters of the prepared matrix gel mixture by mixing gently to avoid forming bubbles.

Keep the tube containing the cells in matrix gel on ice until the mice are ready for the matrix gel injection. To begin, place the 30 gauge one milliliter insulin syringes in a four degrees Celsius refrigerator for cooling. Keep the freshly prepared matrix gel mix containing vascular cells on ice until the mouse is ready for the injection.

Next, place the properly anesthetized mouse on the operating board in a prone position and secure its limbs onto the board using tape. Load a pre-cooled insulin syringe with 300 microliters of the matrix gel mixture, avoiding the formation of bubbles. Disinfect the skin on the back of the mouse using 75%alcohol pads.

Then, subcutaneously inject 300 microliters of the matrix gel mixture in the syringe into one side of the animal's back. Gently remove the needle from the injection site to prevent any leakage of the gel mixture. Ensure the formation of a small hump at the injection site.

Position the mouse on a heating pad for two minutes to allow the matrix gel mixture to coagulate and form a plug. Repeat the demonstrated steps to create a plug on the other side of the mouse's back. Mark the edges of the hump using a marker pen.

House the animal in a specific pathogen-free experimental animal laboratory at 20 to 24 degrees, with 12 hour light and 12 hour dark cycles for seven days. To inject the mouse with dextran FITC seven days post the matrix gel injection, resuspend 0.5 milligrams of dextran FITC in 500 microliters of double distilled water. Thoroughly vortex it to obtain a one microgram per microliter dextran FITC solution.

Fill a 29 gauge syringe with 50 microliters of the dextran FITC solution. Secure the mouse on the tail vein injection instrument. Sanitize the tail with a 75%alcohol-soaked cotton ball before carefully injecting 50 microliters of the dextran FITC solution in the syringe into the tail vein.

Apply pressure to the injection site with the cotton swab for one minute to staunch bleeding. Return the mouse to its cage and let it relax for 30 minutes before collecting the matrix gel plug. To collect the gel plug, cut the skin of the euthanized mouse along the marked border of the plug using surgical scissors and remove the skin above the matrix gel plug.

Collect the plug and rinse it in a beaker with phosphate-buffered saline to wash off any excess blood. Next, cover the bottom of the tissue embedding cassette with about 0.5 milliliters of tissue embedding gel. Then swiftly placed the matrix gel plug on the tissue embedding gel in the planned orientation.

Embed the plug with additional tissue embedding gel. Solidify the cassette at 80 degrees Celsius for 12 hours. After removing the solidified plug block, cut 12 micron thick sections using a freezing microtome.

Then, mount the sections onto microscopes slides.