An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice

We developed a simple Langendorff-free method for isolating individual mouse heart cells by an antegrade packaging technique. This method allows the isolation of heart cells from juvenile to older mice. Langendorff-based retrograde perfusion has been regarded as a gold standard for isolating cardiac myocytes in various experimental animals.

However, cannulation of the LTA is technically difficult in mice due to their small size. For mouse heart harvest, after confirming euthanasia and shaving the abdomen, open the thoracic cavity quickly to expose the heart and use a plastic transfer pipette with the tip cut to approximately the size of the heart to suck the heart into the pipette. Raise the pipette to create enough space to insert curved scissors and use the scissors to excise the heart from the dorsal side taking care to avoid damaging the atria.

Immediately, place the heart into a 30 milliliter glass beaker containing ice cold CIB-EGTA for about one minute. When the contractions have stopped, place the heart in a 35 milliliter culture dish containing ice cold CIB-EGTA, and remove the lung and other visible tissue. Place the roughly cleaned heart onto a heart stand filled with chilled CIB-EGTA apex side down and place the stand under a stereoscopic microscope.

Remove the fat and connective tissues from around the aorta. If the length of the cut aorta is too long, trim the aorta just under the brachiocephalic artery and orient the heart so that the anterior surface is facing forward. Use tweezers to lift the end of the aorta and use a small vascular clamp to clamp the aorta near the atria while gently pushing down on the atria.

Then place the clamped heart on a perfusion plate with the anterior side facing up and hydrate the heart with a few drops of CIB-EGTA. For antegrade perfusion, load a 20 milliliter syringe containing prewarmed CIB-EGTA connected to a flexible extension tube and a marked injection needle onto the infusion pump and start the pump at a 0.5 milliliter per minute flow rate. When the needle and pump have been filled, place the injection needle on the perfusion plate with the shorter side of the diagonal shape in front and slide the needle until it is just touching the apex of the heart.

Carefully, insert the needle near the apex of the left ventricle into the ventricular chamber without twisting or detaching the needle from the plate, watching the mark to estimate the depth of the needle insertion. When the needle insertion is complete, blood should begin to flow from the coronary artery. Use tape to fix the injection needle to the plate and increase the pump speed to one milliliter per minute.

If the heart is perfused successfully, the flow of the buffer in the capillary should be visible just under the epicardium. After two to three milliliters of CIB-EGTA perfusion, replace the perfusion buffer with enzyme mix. After one to two milliliters have been perfused, increase the pump speed to 1.5 milliliters per minute.

Use a pipette to remove the accumulated blood-containing perfusate flowing from the heart as necessary and stop the perfusion when the total volume of perfused enzyme reaches 10 milliliters. At the end of the perfusion, transfer 10 milliliters of enzyme mix from the syringe to a 60 millimeter culture dish on a heater mat and add 20 milligrams of BSA to the dish. Gently swirl the dish to dissolve the powder and remove the injection needle and clamp from the heart.

Remove the ventricles and atria from the heart and place the tissues into the BSA-supplemented enzyme mix. To isolate the ventricular myocytes, use two pairs of tweezers to grasp the epicardium and gently pull the ventricles into small pieces. When all of the ventricle fragments have been generated, disperse the cells approximately 30 times with gentle pipetting and filter the undigested debris through a 100 micron mesh cell strainer into a 15 milliliter centrifuge tube.

After centrifugation, resuspend the cardiomyocyte pellet in prewarmed CIB supplemented with calcium and BSA and incubate the cells for five minutes at 37 degrees Celsius. At the end of the incubation, centrifuge the cells again and resuspend the precipitated cardiomyocytes in an appropriate volume of cell resuspension solution for their maintenance at 37 degrees Celsius until downstream analysis. To isolate the atrial myocytes, transfer the atria to a container of prewarmed CIB supplemented with calcium and BSA and tear the atria into pieces as demonstrated.

Use a 20 microliter pipette set to 10 microliters to disrupt the tissues by pipetting and collect the dissociated cells by centrifugation. Then resuspend the atrial cell in an appropriate volume of cell resuspension solution. In this image, freshly isolated ventricular myocytes can be observed.

This isolation procedure results in a 70 to 80%yield of rod-shaped quiescent ventricular myocytes from eight to 10-week-old mice within roughly five hours of isolation. The ratio of freshly isolated viable cells is lower in mice greater than two years of age. The action potentials recorded in the ventricular and atrial myocytes are similar to those measured in cells obtained by the Langendorff-based method.

Immunostaining analysis can be used to assess the organization of the sarcomeric structure of the ventricular myocytes and the transformation of the cardiac fibroblasts into myofibroblasts after subculture. Western blot analysis is recommended to determine the specific expression of proteins of interest in the atria and ventricles after processing. After perfusion with enzymes, proteins from the atria and ventricles can be easily homogenized in lysis buffer with light force for protein extraction.

It is important to control the direction and the depths of the needle insertion. When inserting the needle into the left ventricle, take care not to pierce the ventricular septum or to penetrate the valve. You can change the composition of the perfusate depending on the purpose of the experiment.

For example, an EGTA supplemented detergent can be used to make a cell-free scaffold of the heart.