This technique is more reliable and efficient and does not require extensive practice or training compared to the most commonly used Langendorff apparatus techniques. The main advantage of this technique is that it limits the ischemic time since the initial perfusion occurs in vivo. While performing this technique, it is essential to avoid bubbles in the manifold and to avoid excessive movement of the heart during digestion, these can decrease the yield.
Demonstrating this technique will be Sarah Sturgill, a graduate student in Dr.Mark Ziolo's laboratory. Begin with clearing the temperature controlled manifold using freshly prepared perfusion buffer. To do so, screw in a 27 gauge needle and clear the bubbles using a luer lock connector.
Ensure no bubbles remain. To isolate the cardiomyocyte, expose the sternum of the fully anesthetized mouse, and lateral to the midline, cut proximally through the ribs and to the axilla. Then cut through the diaphragm, ensuring shallow cuts to avoid the heart.
Clamp the sternum with a hemostat and fold the ribs backward to expose the thoracic cavity. Remove the pericardium from the heart gently before cutting through the inferior vena cava immediately distal to the heart. Inject three milliliters of ice cold clearing buffer into the heart's right ventricle over one minute using a 27 gauge needle.
Next, hold the heart using tweezers. Pull it away from the body and expose as much of the aorta as possible. Then clamp the ascending aorta using a hemostat and excise the heart.
Quickly transfer the clamped heart to the lid of a polypropylene Petri dish containing 10 milliliters of warm perfusion buffer. Place the clamped heart into the supporting platform and inject 10 milliliters of temperature-controlled perfusion buffer at 37 degrees Celsius into the left ventricle over five minutes using a stabilized 27 gauge needle attached to the manifold. Next, transfer the clamped heart and supporting platform to a Petri dish with approximately five milliliters of digestion buffer.
Exchange input syringes for a 50 milliliter syringe containing 25 milliliters of digestion Buffer. Clear the manifold of any bubbles and the remaining perfusion buffer before injection. Carefully replace the needle into the same needle position in the left ventricular apex.
With the help of a perfusion pump, inject 37 degrees Celsius digestion buffer into the left ventricle for 15 minutes using a 50 milliliter syringe. Control the temperature of the solution with a water jacket calibrated to eject 37 degrees Celsius solution. Remove the ventricles from the atria before transferring them to a 10 milliliter beaker.
Add three milliliters of digestion buffer to the beaker and cut the ventricles into large chunks using sharp scissors. Cover the beaker with aluminum foil and place it in a shaking water bath preheated to 37 degrees Celsius for five minutes. After discarding the supernatant, resuspend the tissue chunks in three milliliters of digestion buffer and triturate it for approximately four minutes or until a homogenous mixture is achieved.
Once done, filter the cells into a 50 milliliter polypropylene tube using a 70 micrometer nylon cell strainer. Wash nylon cell strainer with three milliliters of digestion buffer. Return the filtrate to a 14 milliliter round bottom polypropylene tube and centrifuge it.
Discard the supernatant before resuspending the pellet in three milliliters of stop buffer. To the cell suspension, add 54 microliters of 100 millimolar calcium chloride stock to make the final calcium chloride concentration 1.8 millimolar. Let the live cells settle for 10 minutes and remove the supernatant containing dead cells.
After resuspending the pellet in a storage solution, the cells can be used for functional experiments and cultures. Brightfield microscope images of isolated cells showed that the isolation of mouse cardiomyocytes produced rod-shaped quiescent cells with no membrane blebs or rounded edges. The cells showed approximately 80%yield and high survival rates determining the success of the isolation technique.
For successful isolation, it's most important to remove the bubbles from the manifold and insert the needle into the same hole in the heart. Once isolated, the cardiomyocytes can be used for cellular and molecular functional experiments.
