Approaches and Results Neonatal Cells:
The cells are isolated from 1-2 day old pups using the enzymes collagenase (Type II) and pancreatin. After removal from the pups, the hearts are placed in buffer containing the enzymes and gently rocked inside a cell culture incubator. Every 20 minutes, the supernatant is removed to collect the dissociated cells and fresh enzyme buffer is added to the tissue. This procedure is repeated 5-6 times. The cells recovered from each digestion are pooled, filtered, and resuspended in serum-enriched growth media. After 24 hours, the cells are irradiated and serum free culture media is added. Adult Cells:
The cells are isolated by Langendorff perfusion of the heart with a solution containing both collagenase (type II) and protease. The heart is excised from the adult rat or mouse with a short piece of aorta still protuding from the left ventricle. This is then cannulated for retrograde perfusion. The initial perfusate is lightly buffered with EGTA and is intended to rinse completely the coronary circulation and the cardiac chambers of blood. The perfusated is then changed to a low-Ca2+ buffer containing the digestive enzymes. After about 10 minutes, the ventricles are cut away from the rest of the heart and minced and stirred in additional digestion buffer for 2 minutes. The cells are filtered through nylon mesh and centrifuged briefly at 300 rpm. The cells are washed and resuspended twice in solutions containing BSA and higher concentrations of Ca2+ before a final resuspension in DMEM with 10% heat-inactivated FBS. Adult cells that will be cultured are isolated in a similar way but are then aseptically plated onto culture dishes in media containing antibiotics.
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