The overall goal of this procedure is to isolate two discrete populations of multipotent perivascular precursor cells from human cardiac tissue. This method may help answer key questions in the field of cardiac regeneration, such as the contribution made to this process by subsets of the perivascular cell population. The main advantages of this technique are that it results in populations of progenitor cells of reduced heterogeneity and improved viability of a select of major ex-adhesion techniques.
This technique my have implications in the therapy of ischemic heart disease because perivascular stem cell subtypes have been shown to have cardiomyogenic potential. Though this method can provide insight into the role of perivascular cells in cardiac regeneration, it can also be applied to the investigation of other aspects of cardiac disease, such as myocardial fibrosis. Generally, individuals new to this method will struggle because of viable cell use as a result of tissue freshness.
To begin this procedure, after removing the pericardium, major vessels, and atria, place the sample in washing medium in a petri dish. Next, cut the ventricular myocardium into small pieces of approximately one cubic millimeter and process them immediately to obtain the highest cell yield. Then, make fresh digestion solution and warm it to 37 degrees Celsius in a water bath.
After that, filter the suspended tissue pieces through a 100 micron strainer and wash them twice with PBS. Transfer the tissue pieces to a sterile 30 milliliter container with a tightly-sealed lid. Add the digestion solution and place the container within a sealed plastic bag in a 37 degrees Celsius shaking water bath set to 120 RPM.
After 15 minutes, invert the container three times. Then, place it back in the water bath for an additional 15 minutes. Afterward, remove and quench the collagenases with five milliliters of DMEM supplemented with 20%FBS.
Subsequently, triturate the digested tissue by gently pipetting 10 to 20 times with a 10 milliliter serological pipette to break down any tissue clumps. Next, filter the suspension sequentially through 100 micron, 70 micron, and, finally, 40 micron cell strainers to obtain a single cell suspension. Following this, centrifuge the cell suspension at 200 x g for four minutes.
Carefully decant the supernatant and resuspend the pellet in one milliliter of red cell lysing buffer for two minutes at room temperature before diluting it with four milliliters of washing medium. Repeat the centrifugation step and resuspend the pellet in one milliliter of washing medium. Dilute 10 microliters of cell suspension with 90 microliters of washing medium to make a one to 10 dilution of cell suspension.
Mix 10 microliters of the diluted cell suspension with 10 microliters of trypan blue stain and place 10 microliters of the mixture on a standard hemocytometer for cell counting. In this procedure, add 50 microliters of mouse serum to the cell suspension and incubate it at four degrees Celsius for 20 minutes to block nonspecific antibody binding. Next, centrifuge the cell suspension at 200 x g for four minutes.
Remove the supernatant and resuspend it in washing medium. Next, aliquot 50 microliters of the cell suspension into each of the two polystyrene round bottom flow cytometry tubes for the isotype and unstained controls and place the remaining volume into a third tube for multicolor staining. Add five antibodies to the single cell suspension for multicolor staining.
Gently pipette to mix the antibodies with the cells and incubate all the tubes at four degrees Celsius for 20 minutes in the dark. Prepare the compensation control beads by adding one drop of positive beads to 100 microliters of washing medium in five flow cytometry tubes. Add the antibodies individually to each of the five tubes.
Then, incubate all the tubes at four degrees Celsius for 20 minutes in the dark. In the meantime, prepare cell collection tubes, each with 500 microliters of EGM-2 culture medium, and place them at four degrees Celsius. Following antibody incubation, add five milliliters of washing medium to wash the cells and beads in order to remove any unbound antibodies.
Centrifuge all the tubes at 200 x g for four minutes. Repeat the washing and centrifugation steps and carefully decant the supernatant. Then, resuspend the cells in washing medium at a concentration of one times 10 to the 6th cells per 250 microliters.
Afterward, resuspend the beads in 100 microliters of washing medium. Transfer the cell and bead suspensions to the cell sorter on ice in the dark. Add one drop of negative beads to the positive bead compensation control tubes and use these to set the fluorescence compensation.
Then, run the unstained control cells to establish the background fluorescence and set the voltages. Subsequently, run the control isotypes to establish the background fluorescence thresholds related to nonspecific binding. Run the multicolor-stained sample and collect the pericyte and adventitial cell populations into the collection tubes.
In this procedure, add 100 microliters of sterile 0.2%gelatin solution per square centimeter of growth area and agitate manually to coat the entirety of the wells. After that, incubate the plates at four degrees Celsisu for 10 minutes. Then, remove the gelatin solution completely.
Keep the collected cells on ice whilst preparing the culture plates. Next, centrifuge the freshly collected cells at 200 x g for four minutes and gently resuspend the cell pellet in an appropriate amount of EGM-2. Afterward, seed the cells on the gelatin-coated plates.
In this figure, live cells were gated to first exclude CD45-positive cells followed by CD56-positive cells. CD144-positive endothelial cells were then removed from the CD56-negative fraction. From this final population, CD146-positive, CD34-negative pericytes and CD146-negative, CD34-positive adventitial cells were selected and subsequently collected.
Both cultures of FACS-purified myocardial pericytes and adventitial cells exhibit a spindle to stellate cell morphology. Once mastered, this technique will take approximately five hours, if performed properly. While attempting this procedure, it's important to remember that the freshest samples will provide the best yield of viable cells.
Following this procedure, other methods, such as cellular cardiomyoplasty, can be performed in order to answer additional questions, such as suitability of these cells in the treatment of ischemic heart disease.
