The overall goal of this procedure is to isolate pulmonary artery smooth muscle cells from neonatal mice. This is accomplished by first infusing iron containing aeros into the pulmonary vasculature via the right ventricle. The second step is to mince the iron containing lungs into a slurry.
Next, a magnet is used to pull down any iron containing vessels. The final step is to plate the iron containing vessels. The smooth muscle cells then migrate from the iron containing vessels onto the dish.
Ultimately, immunofluorescence for smooth muscle specific markers is used to show that the isolated cell population contains smooth muscle cells. Visual demonstration of this method is critical. Since the iron containing aeros infusion steps are difficult to learn and require practice assisting with demonstrating this procedure will be Kang Lee, a postdoctoral fellow from my laboratory.
To begin collect together all of the necessary equipment for pulmonary artery isolation. When ready, set the PA aros, which contains the iron particles and the lung aros to boil After euthanasia, place the animal on an operating board and sterilize the mouse instruments and gloves with alcohol. With a sterile blade in size, the mouse from the neck to the inferior abdomen to avoid clots in the small vessels of the lungs, exsanguinate the mouse by cutting the renal artery.
After opening the chest cavity, flush three to five milliliters of PBS through the right ventricle until the lungs appear white. Next, expose the trachea through blunt dissection. Once isolated, thread one suture underneath.
Place small forceps underneath the trachea for support, and then insert a 24 gauge angio catheter into the trachea as if placing an iv. Secure the angio catheter with a suture placed earlier.Stir. At this point, take the PA agros out of the boiling water and invert to mix well.
Cool the agros to approximately body temperature by swirling on ice. When properly cooled, inject the PA aros into the right ventricle to perfuse the lungs. The iron particles in the aros mixture will make the lungs appear gray in color.
Proceed slowly as injecting too fast. Might cause the iron to leak into the airways. Next, cool.
The lung aeros down as shown earlier. Draw up the lung aeros and attach the syringe to the 24 gauge angio catheter in the trachea. Infuse the aeros through the trachea to fully inflate the lungs.
Proper inflation aids in mincing the tissue later. Once inflated, remove the heart lung block and submerge it in 25 milliliters of ice cold PBS. To solidify the agarose working in a cell culture hood, place the chilled lungs into the lid of a 10 centimeter tissue culture dish.
Carefully remove the heart thymus, trachea, and any connective tissue from the lungs. Move the isolated lungs to the bottom of the 10 centimeter tissue culture dish and add approximately one milliliter of ice cold, sterile PBS. Next, mince the lungs into very small pieces.
Using two scalpels pulse. Break the tip off of a sterile five milliliter serological pipette, and use this to pipette minced lung slurry into a sterile 50 milliliter conical tube. Use additional sterile ice cold PBS to wash the plate to ensure that all of the lung tissue gets into the 50 milliliter conical tube.
Next, place the 50 milliliter conical tube onto the magnet and wait for the iron containing tissue to move toward it. Depending on the ratio of iron containing vessel to lung tissue, some of the vessels may still be floating at this point. Aspirate off the PBS slowly and carefully trying not to aspirate floating lung tissue since it is unclear if it contains vessels.
Wash the lung pellet three times with five milliliters of sterile PBS, again, trying to minimize the amount of lung tissue that is aspirated. After the final wash, reus suspend the lung pellet in three milliliters of collagenase to avoid large chunks getting stuck in the pipette. Pour the tissue into a 60 millimeter tissue culture dish.
Instead of pipetting. Use an additional three milliliters to rinse the tube, pouring this into the 60 millimeter tissue culture dish as well. Place into a 37 degree Celsius tissue culture incubator.
For one hour after the incubation, pipette the resulting slurry up and down with a 15 gauge blunt needle attached to a five milliliter syringe until all the large lung pieces are disrupted. The tissue plus collagenase slurry should be cloudy with no visible tissue chunks. Pour this into a new sterile 50 milliliter conical tube and attach the magnet.
Wash the tissue culture dish with approximately five milliliters of complete M1 99 media to ensure that all of the tissue has been collected and pour this into the conical tube. Next, aspirate off the collagenase plus media supernatant. There should be very few floaters at this point, and a compact iron containing pellet should be on the side of the conical tube next to the magnet to inactivate.
Wash the pellet with five milliliters of complete media three times. Next, add three milliliters of complete media and mix a few times to resuspend the iron containing pellet. Pour the resuspended pellet into a 35 millimeter tissue culture dish.
Using a microscope, small vessel fragments can be visualized floating around with iron particles contained in the vessel lumen. Finally labeled the dish plate zero and place in a tissue culture incubator overnight as visualized by light microscopy spindle shaped P-A-S-M-C can be seen migrating from the black iron filled pulmonary artery tissue onto the culture dish by day three on day 14. After pooling a population of P-A-S-M-C can be seen on the tissue culture dish.
These images show that P-A-S-M-C stain positive for smooth muscle markers. When imaged at 20 x multiple spindle shaped cells are seen consistent with the smooth muscle cell phenotype. When imaged at higher magnification, the lala podia of the leading edge of single cells are visualized as they migrate and grow towards other smooth muscle cells.
On the dish. Here, phosphodiesterase five activity is shown to be developmentally regulated. The highest levels were isolated from P seven mice with lower levels in P 14 and P 21 mice.
The lowest levels of PDE E five activity were found in adult mice Once mastered. The first day of this procedure can be done in less than three hours if it is performed properly. After watching this video, you should have a good understanding of how to isolate pulmonary artery, smooth muscle cells from neonatal mice ranging in age from P seven to adults.
