As soon as we're in and secured , we excise the heart and remove it to the room in which we perfuse. The perfusion has already begun, of course, as we are actually perfusing as we tie in the cannula, and continue to do so as we transport the heart and hang it on our dog-sized Langendorff apparatus . After the heart is well washed, we insert a plug in the bottom of the effluent chamber, and begin warming, oxygenating and recirculating the buffer that perfuses the heart. We actually lower the heart into the chamber itself, to keep it warm and eliminate loss of buffer. Perfusion time varies from 15 to 30 minutes or more.
Time is less important than feel and appearance in doing a digestive perfusion.
When the heart is soft and beginning to break down significantly, we cut it down from the cannula and slice it into 5 to 7 mm thick cross sections, beginning at the apex. We put the slices in a sidearm flask, add buffer to cover by an inch or so, and shake them in a water bath with constant oxygenation. We pour off buffer every five minutes to collect cells and monitor the progress of the digestion, and add fresh buffer to maintain an appropriate volume. We usually continue digesting for about 15 minutes, then triturate, with a turkey baster, and filter the slurry through cheesecloth, and again through nylon fabric. At this point, it's just a matter of harvesting the cells through a series of spins and washes identical to that which we use with the rabbit cardiomyocytes.