The overall goal of this procedure is to rapidly and completely remove cellular components from an intact porcine heart through retrograde profusion to yield a site-specific cardiac extracellular matrix scaffold. This is accomplished by first cannulating, the aorta of an intact porcine heart. Next tubing is attached to a peristaltic pump, and the tissue is washed with water and salt solutions at low flow rates.
The heart is then perfused with a series of detergent and enzymatic solutions at a gradually increasing flow rate to remove cellular components. The final step is to perfuse a disinfectant solution. Remove the tubing and cut away the ventricular walls for processing.
Ultimately, the heart is analyzed to confirm the complete removal of cellular and nuclear material from the tissue. Visual demonstration of this method is critical because the cannulation and initial profusion steps are difficult to learn because the exact position of the tubing is key to the success of the experiment. The porcine organ is obtained from an abattoir or research facility immediately after euthanasia.
Begin by rinsing off any excess blood. Next, trim the heart of excess fat and tissue, keeping the atria and aorta intact. Separate the pulmonary artery from the aorta by trimming away additional fat.
Once trimmed, wrap each heart individually in freezer paper and store at minus 80 degrees Celsius for at least 24 hours to ensure complete freezing. When ready to use, thaw the heart by submerging it in a four liter beaker filled with type one water and leave it at four degrees Celsius overnight. After the heart is completely thawed, pad it dry and record its weight.
The heart of a market weight pig should weigh approximately 375 to 450 grams. Once the heart is ready, connect size 18 Master flex tubing to the one quarter inch end of a barbed reducer. Next, insert the barbed reducer and tubing inside the aorta.
Place two hose clamps or secure Zip ties around the aorta just below the brachiocephalic trunk. The reducer and tubing must remain above the aortic valve so that the coronary arteries can be perfused. Next, use a syringe to fill the tubing with type one water and insert it into the cartridge of a master flex roller pump at its approximate midpoint.
Next, submerge the inflow end of the tubing, along with the heart in the bottom of a beaker filled with 2.5 liters of water and secure. The most difficult aspects of this procedure are proper cannulation of the heart and maintenance of adequate pump speeds throughout the procedure. We ensure success in our experiment by visually inspecting the tissue for air bubbles throughout the procedure, as well as maintaining proper effusion from the ventricles throughout the procedure.
Prime the pump to remove air bubbles from the tubing, reposition, or secure additional ties around the aorta and tubing if air bubbles are observed. An airtight seal is important to maintain adequate pressure during the decellularization process. At this point, warm a four liter beaker containing three liters of salt balanced trypsin solution in preparation of the decellularization process.
First, set the pump to a flow rate of 400 milliliters per minute and make sure that the correct tubing size is selected. Start the pump and flush the heart with type one water for 15 to 25 minutes, the heart should swell and diffuse blood from the ventricles fresh solution should be substituted every five to 10 minutes or as needed. After the blood is removed, stop the pump and transfer the heart to a separate beaker filled with two XPBS.
Submerge the tubing. Increase the flow rate to 700 milliliters per minute and start the pump. Leave the heart in solution for 15 minutes, changing the solution every five minutes.
When ready, transfer the heart to type one water for 10 minutes and increase the flow rate to 750 milliliters per minute. To begin the decellularization process, transfer the heart to the beaker containing warm salt balance tryin solution. Increase the speed to 1200 milliliters per minute and start the pump.
Place a stir bar at the bottom of the beaker for circulation and leave the heart in this solution for three hours. Perfusion speeds are increased slowly to condition the tissue and prevent rupture of the vessels. After one hour, increase the pump speed to 1500 milliliters per minute.
After an additional hour, the pump speed is increased to 1800 milliliters per minute. The heart should swell and nearly double in size. The tissue should lose its natural color progressing from the atria to the apex.
After each solution perfusion, a two step rinse is performed to remove cellular debris, chemical residue, and aid cell lysis After the salt balanced trips and solution, perfuse water at 1900 milliliters per minute and then two XPBS at 1950 milliliters per minute for 10 minutes each. Next, transfer the heart to a room temperature salt balance solution of Triton X 100. Increase the pump speed to 2000 milliliters per minute and perfuse for one hour.
After refilling with fresh solution, increase the pump speed to 2100 milliliters per minute and perfuse for an additional hour and a half. Next, rinse the tissue as demonstrated before, but at increased pump speeds. The next perfusion is with room temperature 4%sodium deoxy coate.
Increase the pump speed to 2200 milliliters per minute and perfuse for three hours. After three hours, repeat the rinsing steps, changing the solutions after five to 10 minutes for each solution. After the final perfusion, transfer the heart to a 0.1%paracetic acid, 4%ethanol solution and perfuse for 1.5 hours at 2200 milliliters per minute at the same pump speed, perfuse the tissue with one XPBS for 15 minutes, followed by two five minute washes in type one water.
Repeat once more to complete the solution perfusion procedure, turn the pump off and remove the heart from the solution. Cut the ties from the aorta. Remove all tubing and place the heart in an empty beaker to drain for one hour.
Remove excess liquid periodically after one hour. Lay the heart on an absorbent pad to drain further. After most of the water is removed, record the final weight of the heart.
The heart can lose approximately 20 to 25%of its initial weight during the decellularization process. At the end of this process, the heart should appear white or translucent indicating the loss of cellular material. Next, the ventricular septum and right and left ventricles are separated and reserved for DNA.
Quantification and histological processing freeze the samples at minus 80 degrees Celsius for at least two hours before lyophilization. Successful decellularization is demonstrated here by the significant decrease in DNA content when compared to native ventricles. This gel shows that little residual DNA remained in the decellularized ventricles when compared to a urinary bladder matrix or UBM.
In this example, hemat toin and eosin staining shows the complete removal of nuclear material from the ventricles. Masson's staining further confirms the loss of cardiac muscle bundles and the retention of collagen networks While attempting this procedure. It is important to visually inspect the tissue and the coronaries frequently.
