Here, we present a detailed protocol for isolating endocardial cell and coronary endothelial cells separately from the heart to help people understand the cell type-specific gene expression and function. The main advantages of this method are high purity and viability of the isolated EEC and CECs and that the cells maintain their physiological properties upon isolation. CECs and EECs differ in many aspects, so the ability to independently investigate these cell populations enables the exploration of cell type-specific mechanisms in various heart diseases.
This method could facilitate the study of transcriptonomic and epigenetic factors responsible for numerous heart diseases in a cell type-specific manner. The trickiest parts of this method are determining the different heart portions and timing the digestion correctly to avoid cell type contamination. Demonstrating the procedure will be Yifei Miao, a research scientist from my lab.
After harvesting hearts from six 50 to 100 gram Sprague Dawley rats, wash the organs with 50 milliliters of cold HBSS three times to remove any excess blood and place one heart under a dissecting microscope. Position the heart on its flatter posterior face to identify the left and right sides of the tissue and locate the pulmonary artery. Use scissors to cut through the pulmonary artery down to the right ventricular chamber and cut along the septum until the apex is reached.
Continue cutting from the apex up the posterior side of the heart along the septum until the junction of the pulmonary artery and the right ventricular chamber is reached. Starting from this point, cut both the anterior and posterior sides of the heart perpendicularly to the previous dissection point and away from the septum until the right ventricular free wall is liberated from the rest of the heart. Then place the right ventricular free wall into a five milliliter tube of DMEM and locate the aorta again to facilitate collection of the left ventricular free wall in the same manner as just demonstrated.
When all of the ventricular free walls have been collected, transfer the tissue samples into a 60 centimeter culture dish with their inner surfaces lying face down in the dish. Using a one milliliter pipette tip, add 0.5 to one milliliter of digestion buffer to the dish directly under the tissue pieces until only the inner surfaces of the tissues are immersed. To avoid undesired cell contamination, it's important that only the inner surface of the ventricle is immersed in the digestion buffer and it's digested for exactly five minutes.
After five minutes in the cell culture incubator, stop the digestion with five times the volume of endothelial cell medium. Then use a one milliliter pipette to flush the inner surface of each ventricle with fresh medium transferring the runoff through a 40 micrometer strainer into a 50 milliliter collection tube on ice. For coronary endothelial cell digestion, cut along the outer surface of the left ventricle without contamination from the inner layer and place each tube fragment in a separate five milliliter tube containing one milliliter of digestion buffer.
To avoid contamination from EECs, it's important to only cut from the outer surface of the ventricle. Using dissection scissors, mince the ventricular wall into small one cubic millimeter pieces and place the tubes in a 37 degree Celsius water bath for 15 to 20 minutes with vortexing every two to three minutes. When the minced pieces are small but visible, add four milliliters of endothelial cell medium to the tube and use a five milliliter serological pipette to transfer the entire volume of solution through a 40 micrometer strainer into a 50 milliliter collection tube on ice.
To isolate the CD31 positive cell populations after their digestion, pellet the cells by centrifugation and aspirate the supernatants. If any pellets are red, resuspend the cells in one to two milliliters of red blood cell lysis buffer per tube for a five-minute incubation in a 37 degree Celsius water bath. At the end of the incubation, stop the lysis with 10 milliliters of PBS and collect the cells with a second centrifugation.
Next, add 90 microliters of sorting buffer and 10 microliters of anti-CD31 PE antibody to each tube of cells. After vortexing, incubate the cells for 10 minutes at four degrees Celsius. At the end of the incubation, add 10 milliliters of sorting buffer to the tubes with thorough mixing and collect the cells by centrifugation.
Resuspend the pellets in 80 microliters of sorting buffer per tube, followed by the addition of 20 microliters of anti-PE microbeads. After vortexing, place the tubes at four degrees Celsius for 15 minutes, followed by washing in 10 milliliters of sorting buffer by centrifugation as just demonstrated. Resuspend the pellets in 500 microliters of sorting buffer on ice and place a column containing super paramagnetic spheres into a magnetic separator.
Rinse the column with three milliliters of sorting buffer. When the column has emptied, add 500 microliters of the endocardial endothelial cell suspension to the column, followed by four washes with three milliliters of sorting buffer per wash. After the last wash, transfer the column into a new 15 milliliter collection tube and use the column plunger and five milliliters of fresh endothelial cell medium to flush the CD31 positive cells out of the column into the collection tube.
Then collect the bead isolated endothelial cells by centrifugation. As predicted, quantitative PCR reveals that relative to beta actin, endocardial endothelial cells expressed higher levels of the endocardial markers Npr3, Hapln1, and Cdn11 compared to coronary endothelial cells. Likewise, coronary endothelial cells expressed higher levels of the coronary markers Fabp4, Mgll, and Cd36 compared to endocardial endothelial cells.
Additionally, both types of cells expressed the pan-endothelial cell marker gene Cdh5 with slightly higher levels observed in coronary endothelial cells. We can further purify the cells using FACS and antibodies specific to the cell populations. For example, we can use anti-Npr3 to sort EECs and anti-Cd36 to sort CECs.
This technique paved the way for researchers to explore the role of EEC and CECs in heart development and disease in a cell type-specific manner.
