Isolation of high quality cardiac myocytes is key to the successful experiments with fresh or cultured cells. Though, the isolation of myocyte has been conducted for several decades, there remains no single easy method that can produce a large yield of high quality, viable cells without adjustments. Researchers may face many difficulties during the isolation process due to the factors such as poor water quality, unclean or contaminated glassware and instruments, incorrect pH or temperature of solutions and etc. Here are some most prominent factors regarding to the isolation of cardiomyocyte from animal hearts.
- Preparation of the Langendorff apparatus
Most scientists believe that high quality cardiomyocytes can be more reproducibly isolated by the Langendorff method, using retrograde perfusion through the aorta with enzyme-containing solutions. In a basic Langendorff apparatus, tubing leading from the solution reservoirs leads to a cannula, on which the heart is mounted via the aorta. The size of the cannula must be carefully chosen based on the size of the aorta.
- Preparation of the animals
Animals used for cell isolation must be handled gently to minimize stress that may affect the neuro-humoral state of the cells. It is vitally important to inject these animals with heparin (400–5000 u/kg b.w.) 20–30 minutes before the anesthetization to prevent blood clotting and possible myocardial infarction. The anesthetization by using either injectable or inhalable anesthetic is needed, for it can help avoid the risk of myocardial ischemia of small animals such as mice and rat.
- Dissection and cannulation
The researchers need be more careful during the excision, because less tissue around the heart makes it easier to find and clean the aorta. And they need to leave as much of the ascending aorta as possible to facilitate cannulation. Then the heart should be rapidly immersed in cold nominally Ca2+-free solution and the area surrounding the aorta should be cleaned of excess tissue. After the excision, the aorta will be positioned onto the cannula using fine-tipped forceps and secured with a silk suture (size 4-0). To make cannulation easier, the use of a dissecting microscope or magnifying glasses is recommended. The cannula should be carefully removed from the syringe and fastened to the Langendorff setup to avoid introducing air bubbles.
- Using proper enzyme for perfusion
The perfusion of cell isolation usually starts with a zero or low Ca2+ solution, to disrupt intercellular connections at the intercalated discs, followed by enzyme digestion to break down the extracellular matrix. The time of perfusion with enzyme solution is either experimentally established and used consistently for each species, depending on the heart weight of each animal, or determined by palpation. After perfusion, the heart should be soft to the touch, flaccid and pale.
After perfusion is completed, the heart will be taken down from the cannula and, depending on the investigators’ goals for experiments; the ventricles and/or atria need to be dissected and put into the proper solution. The preparation is cut into small pieces and gently triturated with a plastic transfer pipette. It is important to use pipettes with a relatively large opening and no sharp edges to minimize mechanical stress and cell tearing during trituration.
- Sedimentation of cells
To enrich the cell suspension with viable cardiac myocytes and to remove large, undigested chunks of tissue, dispersed myocytes need to be filtered though a mesh cell collector. Cells are able to sediment either by gravity or gentle centrifugation, which allows the separation of the rod shaped cardiac myocytes from other cell types present in the heart and from rounded dead myocytes. The sedimentation should be repeated several times, while Ca2+ concentration is gradually increased in several steps. This allows the cells to gradually return to normal cytosolic Ca2+ levels without becoming Ca2+ overloaded and depolarized.
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