Isolation of Swine Cardiomyocytes
Christy Shivell and
Charles Ganote
East Tennessee State University, Department of Pathology
PO Box 70568
Johnson City, TN 37614
Phone (423) 439 - 7018
Shivell@access.etsu.edu
The isolation of cardiomyocytes from swine is not a project to be undertaken lightly, but the investigator who diligently pursues this goal will be rewarded with beautiful cells, a high percentage of rods, and excellent total yield. Working with a large animal such as the pig involves entirely different logistics than are required for small animals. The major hurdles to overcome are cost, personnel, and space and equipment requirements.
EXPENSE
In 1996, we estimated our total cost per pig to be just over five hundred dollars, including all expenses with the exception of man hours. (We made this estimate when pork prices were low.) When you are just starting out, you can expect to have at least one or two flops, making your initial few good preps all the more expensive to obtain. However, once we became proficient with the procedure, we never had a bad preparation, and we found that the high cell yield actually brought the "cost per viable cell" more in line with that of our rabbit model.
PERSONNEL
Several skilled people are necessary to make the isolation run smoothly. A veterinarian or veterinary technician should be on hand to monitor the pig and maintain anesthesia. A skilled large animal surgeon and an assistant are needed to open the chest, cannulate the aorta, and excise the heart. Two people are needed to perfuse the heart, although one could do it in a pinch. You will get an almost overwhelming volume of cells, needing to be filtered and spun and respun, and then you will have enough cells to run multiple experiments, if you have enough people to run them.
In our situation, our division of laboratory animal resources is instrumental to our work with swine; they do all the pre-operative preparation, and provide the necessary veterinary support during the surgery. They also dispose of the carcass properly. Dr. Race Kao, of our Department of Surgery, provides his excellent surgical skills, and the copious amounts of surgical tools necessary. I assist Dr. Kao, and Dr. Stephen Armstrong is on hand to assist further. After the heart is removed, Dr. Armstrong and I remove it to another room, to continue the perfusion and post-perfusion processing to obtain isolated cells. There are sufficient cells to form all the experimental groups the lab can handle.
SPACE AND EQUIPMENT REQUIREMENTS
As for space, you will need at the minimum, a dedicated surgical area, and an area to perfuse, where you can perform a gravity driven perfusion (requires about five to six vertical feet, and three horizontal, minimally), that can get quite messy. Our space concerns are also addressed by Dr. Kao, who provides us his surgical suite, and the room in which we do the perfusion. He has a permanent framework of bars and clamps, pumps and water baths and such, that makes it easy for us to come in and make slight, reversible modifications to do our perfusion.
If you have isolated cardiomyocytes from other species, and even if you've not, you will have most of the tools and equipment that you need. Big -ticket and / or indispensable items include a recirculating water bath, a pump, and a centrifuge. We use a modified Langendorff apparatus during the perfusion, but the same effect could be obtained by putting a catch basin in a water bath. Your surgeon should have all the rest of what you need, plus any tools that he needs for the surgical procedure. You will probably have to make your own cannula. We use a length of tubing (TYGON S-50-HL, Class VI, 3/8 x 1/16), with a beveled piece of aortic cannula (approximately 12mm OD, 7 mm ID, walls 2+ mm thick) attached to one end, and a bifurcated back end that allows us to hook up two iv bags at once and accomplish smooth transitions from one to the next as we perfuse. The entire cannula is one meter long, not including the iv bags. You'll need five of these bags per experiment. Your surgeon can probably help you locate some spent bags that only had ringers or saline or the like in them. You'll also need a good SHARP knife to slice up the heart when going from perfusion to post-perfusion digestion.
BUFFERING
Having surmounted these hurdles, you must then consider buffering, and other details. Our recipes are attached. We initially tried to buffer as we do with rabbits, but were not able to obtain satisfactory results consistently until we began buffering with Hepes throughout the procedure. We filter all our buffers, pumping them into their respective iv bags as we do so. We use a sterile mini capsule filter, from Gelman Sciences, pore size 0.45 µm. The bags are then oxygenated and kept in our water bath, set at 37° C. (This is the same water bath that warms our effluent/recirculation chamber, and in which we shake, by hand, our heart during the post-perfusion digestion.). We don't wet and filter the collagenase (Worthington Type II, CLS2) until we are ready to add it to begin the digestive phase of the perfusion.
CANNULATION AND EXCISION
This phase of the isolation of cardiomyocytes is really too involved to cover in any real detail in this format. Simply put, we perform a thoracotomy, and then cannulate the thoracic aorta, advance the cannula into the ascending aorta, and position the tip of the cannula just distal to the aortic valve. The cannula is tied in tightly to eliminate leakage, and also to prevent the heart from slipping off during the perfusion. We find that umbilical tape works far better for making these ties than any kind of suture material. 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.
PERFUSION
Buffer pours off the heart into the inner chamber of the Langendorff apparatus during the perfusion. We run two liters of heparinized buffer through each heart, and then follow with one or two liters of the same buffer without heparin, depending on how "clean" the effluent appears. The flow rate is entirely gravity driven, and in the early stages of perfusion equals about 100 ml/min, with a pressure of roughly 80 mm Hg. 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. We wet, filter, and add the collagenase at this time, using 75 units/ml. Perfusion time varies from 15 to 30 minutes or more. Time is less important than feel and appearance in doing a digestive perfusion.
POST-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. (This is the very buffer we used in the perfusion, we collect it after we cut down the heart.) 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. Finally, after all your hard work, your rods, perfectly lovely in Ca free buffer, may square and die upon even incremental calcium additions. We solved this problem by becoming proficient with the isolation procedure and by implementing buffering adaptations, which are reflected in our recipes.
A FEW PARTING WORDS
Swine cardiomyocytes are worth the trouble if you can obtain them. It may not have been possible for us to be successful had we not been availed the expertise and support of Dr. Kao, and I strongly discourage anyone from pursuing this avenue of research without the inclusion of someone who has significant large animal surgery experience. You will need his or her skills and surgical equipment to be successful. Feel free to call or e-mail to ask questions or discuss problems.
RECIPES
LOW POTASSIUM KREBS-HENSELEIT
BUFFER, modified
125 mM NaCl
30 mM Hepes
1.2 mM KH2PO4
4.75 mM KCl
1.2 mM MgSO4
Make up four liters and add 7.8 g dextrose to it. Heparinize two liters of the buffer (1000 units/L) adjust pH to 7.5, filter, put in bag, and warm to 37°C.
PERFUSION SOLUTION
58.5 mM Taurine
24.9 mM Creatine
10.8 mM Dextrose
0.1% BSA
1% BME Amino Acids
1% MEM Non-Essential Amino Acids
0.67 mM Glutamine
125 mM NaCl
1.2 mM KH2PO4
30 mM Hepes
4.75 mM KCl
1.2 mM MgSO4
Adjust pH to 7.5, filter, place in bag bag and warm to 37°C. Use a small portion, 100 ml?, to wet and filter collagenase. flush filter with 100ml? more to get as much collagenase out as possible, then add all back to the Langendorff to recirculate. You'll use about 500 mg collagenase / liter, depending on the strength of the lot you have.
SYNOPSIS
Adult pigs (20-30 kg) are injected with Acepromazine (1.0 mg / kg) and atropine (1ml / 10kg), then with ketamine (15-20 mg / kg). They are then anesthetized with pentobarbital (5 to 10 mg/kg) and undergo tracheal intubation and lactated ringers infusion. Anesthesia is subsequently maintained with pentobarbital (5 - 15 mg/kg/hr as required). A thoracotomy is performed after which the aorta is cannulated and the heart excised.and perfused with Hepes buffered saline (30 mM Hepes, 125 mM NaCl, 1.2 mM KH2PO4, 4.75 mM KCl, and 1.2 mM MgSO4) with glucose (10.8mM) and Heparin (1000 units / L) added. The heart is perfused with 2 liters of this buffer after which it may be perfused with 2 more liters of the same buffer minus heparin, if continued washout is deemed necessary due to persistent blood in the system. After the washout phase is complete, the heart is perfused in a recirculating manner on a gravity driven system with the same buffer, non-heparinized,with further additions of creatine (24.9 mM), taurine (58.5 mM), Bovine Serum Albumin (0.1%), vitamins (1%), BME amino acids (1%), MEM non-essential amino acids (1%) glutamine, and EGTA (0.025mM). After one minute, Collagenase (75 units / ml, Worthington Type II) is added, and perfusion is continued until the heart begins to show signs of digestion. The heart is then removed from the perfusion apparatus, sliced, triturated, and incubated in a water bath at 37° C with Oxygen for 15 minutes. The resultant slurry is filtered through cheesecloth and again through nylon fabric, and the cells therein are harvested through a series of spins and washes.