The overall goal of this cell isolation procedure and migration protocol is to show a reliable way of isolating endothelial progenitor cells and their migratory potential towards serum samples of cardiac surgical patients. This method can help to answer key questions in the regeneration of the endothelial lining and blood vessels, which is needed after cardiac surgery due to ischemia and re-perfusion related injuries. The main advantage of this technique is the relatively simple way of isolating progenitor cells, including the pre-isolation of CD-34 positive cells.
In addition to deaths, major complications of the cardiac surgery remain too common. Underlining the need to identify the tie risk and protective mechanisms during cardiac surgery. Endothelial progenitor cells are crucially involved in the neovascularization of ischemic tissues and known to provide cardio-protective properties.
To begin the experiment, mix blood, one-to-one, with calcium free and magnesium free PBS. Add 15 milliliters of density gradient solution to a 50 milliliter tube. And slowly layer the diluted blood on top of the density gradient solution.
Next, centrifuge the samples. Using a sterile plastic pipette, carefully collect the buffy coat layer of every tube, and place it into another tube while avoiding the density gradient solution. Dilute the peripheral blood mononuclear cell, or PBMC fraction, with at least three volumes of PBS and mix the solution by pipetting.
Centrifuge the mixture at room temperature for 15 minutes at 200 times G.Then, aspirate the supernatant, and resuspend the cell pellet in five milliliters of endothelial cell growth medium, MV-2. After resuspending the cells, add 100 microliters of human CD-34 anti-body per used buffy coat and rotate the cells. Add 50 microliters of Dextran coated magnetic beads per used buffy coat and rotate the cells.
After incubation, transfer the suspension to fax tubes with a maximum of three milliliters to each tube. Next, insert the fax tubes into the magnets and wait for five minutes. Discard the super natant without pulling the fax tubes out of the magnet.
Then, outside of the magnets, resuspend the cells in each fax tube with three milliliters of MV-2 medium. Transfer the cell suspension into pre-coated T-75 flasks. Add 17 milliliters of MV-2 medium to each flask.
To prepare the migration assay, use a pipette to remove the medium from the endothelial progenitor cells or the EPCs, in the T-75 flask. Wash the cells with five milliliters of phosphate buffered saline or PBS, and carefully shake the flask. Next, remove the PBS and add five milliliters of commercial cell detachment solution.
Then, wait until the cells are detached under a light microscope. Accelerate the detachment by carefully tapping the bottom of the flask. When the cells are detached, quickly add five milliliters of MV-2 complete medium, and transfer the cell suspension into another tube.
Centrifuge the cells at 2, 000 x G for five minutes. After pelleting the cells, resuspend them in five to ten milliliters of PBS. And centrifuge them again.
Resuspend the cells in five to ten milliliters of PBS again and follow with centrifugation. Resuspend the cell pellet in MV-2 starved medium. Next, dilute the serum sample one to five in MV-2 starved medium.
Prepare the migration plate by adding 235 microliters of the serum sample into the lower chamber. Add the insert shortly before adding the cell solution. Then, add 75 microliters of the cell solution into the upper chamber.
Allow the EPCs to migrate. Remove the upper chamber containing all of the non-migrated cells. Add 75 microliters of 3.6 percent paraformaldehyde solution including hoechst dye diluted at one to 1, 000.
Centrifuge the plate shortly to get all of the cells into the same focal plane at 2, 000 x G for one to two minutes. To avoid serum auto-fluorescence artifacts, quantify the migrated cells by taking five pictures per well with 100x magnification under a microscope. Finally, count the migrated cells using the semi-automated software.
Fax analysis of the EPCs verify the uptake of acLDL, as well as the expression of CD-31 on the surface of the isolated cell population. Isolating the analysis of acLDL and CD-31 reveals a homogenous distribution of each marker. Elyso to identify the influence of cardiac surgery following myocardial reperfusion injury on the concentrations of circulating serum levels of MIF, CXCL12, CXCL8, and VEGF.
Serum samples were drawn pre and intraoperatively. Serum levels of MIF, CXCL12 and CXCL8 showed a significant intraoperative increase compared to baseline values. In contrast, VEGF concentrations did not show any significant changes.
An ex vivo migration assay, using serum samples drawn prior to and during surgery was performed using EPCs isolated from healthy volunteers, and revealed a significantly increased migration rate toward the intraoperatively taken samples. Following this technique, peripheral blood mononuclear cells can easily be isolated to, in order to answer additional inflammatory related questions.
