マウス冠動脈血管平滑筋細胞の単離

The overall goal of this procedure is to isolate vascular smooth muscle cells from the murine coronary circulation for their culture and use in standard cell culture assays. This method can help answer key questions in the cardiovascular field such as what key disease mechanisms are involved in murine primary coronary vascular smooth muscle cell behavior. The main advantage of this technique is that it yields a mostly pure population of mouse primary coronary vascular smooth muscle cells.

Before beginning the procedure attach a sterile 10-milliliter Leur Lock syringe filled with room temperature HBSS without phenol red to the perfusion pump tubing. Next, transfer each of the 25-gauge cannulas from the heating coils onto individual 10-milliliter syringes filled with ice-cold HBSS without phenol red. Place the syringes on ice.

Then, tie loose, overhand knots in each of two five-centimeter pieces of silk suture with one end twice as long as the other. Place one knot onto each of the 25-gauge cannulas without tightening and place one of the HBSS-filed syringes into a burette clamp on a ring stand. When all of the materials are ready, confirm a lack of response to toe touch and use scissors to remove the fur and skin on the anterior neck of the anesthetized experimental mouse.

When the jugular vein is visible use forceps to carefully remove any fat surrounding the vein and slowly inject 100 microliters of heparin, placing a surgical sponge over the vein to avoid excessive bleeding. After one minute, open the chest to expose the heart. Using curved forceps to life the heart, excise the organ with scissors, taking care to keep the ascending aorta intact through the first brachiocephalic branch.

Then, immediately rinse the heart in a 35-millimeter dish containing ice-cold HBSS without phenol red. Next, position the ring stand-mounted cannula at a 45-degree angle next to a dissecting microscope so that the tip is just under the surface of a second petri dish of ice-old HBSS and clearly visible through the objective. Transfer the heart under the microscope and use Dumont forceps to gently blunt dissect the excess adipose tissue to prevent the puncturing or tearing of the aorta.

When all of the tissue has been removed, use forceps to slide the vessel over the cannula and guide the needle downward through the aortic lumen close to but not beyond the aortic valve. Once the cannula is in place, slide the pre-tied silk over the aorta and tighten the knot. Then, tie a second knot to form a square knot around the aorta, securing the heart.

Now, slowly and gently flush the remaining blood from the coronary circulation with the ice-cold HBSS and turn on the perfusion pump to begin the flow of HBSS. Remove the cannula and heart from the syringe, taking care to keep the cannula hub filled with HBSS and connect the cannula to the warm HBSS-filled heating coils. Then, begin an eight-minute HBSS perfusion of the heart at a rate of 0.5 milliliters per minute.

Immediately cannulating and perfusing the next heart as soon as the perfusion of the first heart has begun. After all of the HBSS has been perfused, replace the HBSS syringe with 10 milliliters of pre-warmed digestion solution and perfuse the hearts with the digestion solution at 0.5 milliliters per minute. After 12 minutes change the perfusion rate to 0.4 milliliters per minute and place 15-milliliter conical tubes filled with cold stop solution into an ice bucket beneath the hearts to begin the vascular smooth muscle cell-enriched perfusate collection.

After 15 minutes replace the first set of collection tubes with a second set of tubes and immediately spin down both tubes containing the first vascular smooth muscle cell fraction. When less than one milliliter of digestion solution is left in the syringe, attach a new syringe containing 10 milliliters of fresh digestion solution. At the end of the centrifugation aspirate all but the last 0.5 milliliters of supernatant from each collection tube and resuspend the pellet from the first heart in two milliliters of warm stop solution.

Next, mix the cell suspension from the first heart with the pellet from the second heart and store the pooled cells in a 37 degrees Celsius incubator with a loose cap. After 80 minutes of digestion, cut open the heart apex to flush out any of the remaining cells trapped inside the ventricle and collect these cells in the sixth set of collection tubes. When all of the cell fractions have been combined, spin down the pooled vascular smooth muscle cell rich suspensions and aspirate as much of the supernatant from the collection tubes from the two hearts as possible.

Then, resuspend the pellets in two milliliters of plating medium and pool them in a 35-millimeter culture dish at 37 degrees Celsius for 24 hours of culture. The next day, gently aspirate the medium from the vascular smooth muscle cell culture in a biosafety cabinet. Then, wash the cells two times with two milliliters of warm sterile PBS gently tapping the dish against two fingers while rotating the plate in a clockwise motion for 360 degrees.

After the second rinse, replace the wash with two milliliters of fresh warm sterile PBS and check the culture under the microscope to confirm confluency and the absence of debris. Replace the PBS with two milliliters of warm plating medium and return the cells to the incubator for 24 hours. The next day, wash the cells again to remove any final debris and dead cells from the culture.

Then, replace the PBS with two milliliters of warm plating medium and return the cells to the incubator. As assessed by the morphology of the cells in the culture after the first wash the isolation procedure effectively removes cardiac myocytes and endothelial cells. To rule out the possibility of contamination by adventitial and interstitial fibroblasts, the isolated cells can be stained for SM22 alpha and alpha smooth muscle actin, both vascular smooth muscle cell markers, and vimentin, a fibroblast marker up through passage two.

Staining for PCAM also confirms the lack of endothelial cell contamination, demonstrating that a nearly pure population of vascular smooth muscle cells can be isolated from the coronary circulation by this protocol. Once mastered, this entire technique can be completed in less than two hours if it is performed properly. After watching this video you should have a good understanding of how to isolate primary murine vascular smooth muscle cells.