The overall goal of this procedure is to simultaneously isolate viable cardiomyocytes, endothelial cells, and fibroblasts from the rat heart for their individual in vitro analyses. This method can help answer key questions in the cardiovascular field about the signaling mechanisms involved in cardiac hypertrophy, ischemia reperfusion injury, endothelial function, and cardiac fibrosis. The main advantage of this technique is that all of the major cardiac cells can be isolated simultaneously, potentially reducing the associated research costs and experimental animal numbers.
After one distilled water and one 50 milliliter Powell medium flush, replace the medium with 80 milliliters of fresh Powell medium, and use a glass Pasteur pipette from the glass cylinder to continuously perfuse the medium with carbogen. Next, use two pairs of thin curved forceps to mount a freshly harvested rat heart via the aorta onto the Langendorff perfusion system, and place the end of the perfusion system cannula between the first aortic branch and the aortic valve. Fix the aorta with a crocodile clamp.
Open the valve of the cannula, and then fix the heart with a surgical suture. Allow 35 milliliters of the perfusion medium to pass through the heart drop wise, removing any residual blood. Place the collecting funnel under the heart, and start the peristaltic pump to recirculate the perfusion medium from the collecting funnel to the reservoir.
Add collagenase solution to the perfusion medium, and perfuse the heart with the recirculating collagenase solution for 30 minutes. At the end of the digestion, use scissors to remove the heart from the Langendorff system and place it in a glass Petri dish. Now remove both atria and any leftover fatty tissue from the heart.
To avoid epithelial cell contamination, use two forceps to carefully peel away the pericardium. Cut the heart down the middle, and place the halves into the tissue chopper. Mince the heart two to three times, and transfer the tissue pieces into a 15 milliliter conical tube containing 12 milliliters of digestion buffer from the Langendorff perfusion.
Digest the tissue fragments in a water bath for five to 10 minutes, occasionally mixing with a five milliliter disposable plastic pipette. Then filter the cell suspension through a 100 micrometer sieve into a 50 milliliter conical tube. Flush the perfusion system with a minimum of one liter of distilled water followed by 100 milliliters of 0.1 normal sodium hydroxide for 30 minutes, then flush the system with another two to three liters of distilled water, and dry the apparatus with flushed air.
Collect the filtered cells by centrifugation, and use a disposable pipette to carefully transfer the endothelial cell and fibroblast containing supernatant into a new 50 milliliter tube. Resuspend the pellet in six milliliters of calcium solution one. After one minute, collect the cells with a second centrifugation, and resuspend the pellet in six milliliters of calcium solution two.
After one minute, dilute the cell suspension with the addition of 12 milliliters of calcium solution three, and mix well by gentle tilting. After another centrifugation, resuspend the pellet in 20 milliliters of pre-warmed CCT medium. Then aliquot one milliliter of cells to each of 20 sterile 35 millimeter cell culture dishes pre-coated with laminin, and place the cells in a carbon dioxide free cell culture incubator, replacing the medium with two milliliters of fresh CCT medium to remove any dead cells after two hours.
Now centrifuge the endothelial cell and fibroblast containing supernatant, and resuspend the pellet in 1.5 milliliters of endothelial cell isolation buffer. Transfer the cells to a two milliliter sample tube, and add mouse anti-rat CD31 antibody to the cells for a 30 minute incubation at four degrees Celsius with end to end rotation. At the end of the incubation, add Pan anti-mouse IGG beads for a 20 in incubation at four degrees Celsius, with end to end rotation.
At the end of the bead incubation, place the cells in a magnetic rack for two minutes, and use a one milliliters pipette to carefully remove the mostly fibroblast containing supernatant. See the fibroblasts on a 10 centimeter culture dish containing 10 milliliters of fibroblast cell culture medium, and place the cells in a cell culture incubator at 37 degrees Celsius and 5%carbon dioxide for one hour. Next, add one milliliter of wash buffer to the tube of endothelial cells and cap the tube.
Gently shake the cells four to five times to resuspend the beads. Then return the tube to the magnet, and remove the wash buffer after one minute. After the last wash, replace the wash buffer with one milliliter of MV2 endothelial cell culture medium, and seed the endothelial cells into a single well of a 12 well plate, for their overnight culture in the cell culture incubator.
At the end of the fibroblast incubation, wash the attached cells with an appropriate volume of pre-warmed PBS two to three times. Then, add pre-warmed fresh fibroblast cell culture medium to the cells, and return the fibroblasts to the cell culture incubator. The next morning, replace the supernatant on the endothelial cell culture with fresh endothelial cell culture medium, and return the cells to the incubator, replacing the medium every two to three days, until semi-confluency.
Then wash the culture of attached fibroblast cells with fresh pre-warmed PBS as just demonstrated, and feed the cells with fresh pre-warmed medium. The cardiomyocyte isolation procedure yields a 70 to 80%pure, viable, rod-shaped, striated cardiac cell population. Intracellular calcium oscillation analysis of isolated cardiomyocytes loaded with fura AM, in response to ischemia repurfusion in cardiomyocytes can then be performed, as well as analysis of the changes in calcium signaling in magnetic bead isolated endothelial cells and fibroblasts, after the addition of ATP.
Once mastered, this technique can be completed in three hours if it performed properly. While attempting this procedure, it is important to remember to set up this perfusion system properly, and to promptly fix the heart in the system as soon as it is ready. After it's development, this technique paved the way for researchers in the field of cardiovascular research to explore the signaling involved in different cardiovascular pathophysiologies.
After watching this video, you should have a good understanding of the basic principals of cardiac cell isolation and culture. Don't forget that working with surgical instruments and cell culture reagents can be extremely hazardous, and that precautions, such as careful usage of the instruments and the use of gloves should always be taken while performing this procedure.
