Purification of CD31⁺ Endothelial Precursor Cells Using Magnetic-Activated Cell Sorting

Take an adherent culture of endothelial progenitor cells or EPCs, precursors to brain microvascular endothelial cells.

The culture, obtained by differentiating pluripotent stem cells, comprises EPCs positive for the surface marker CD31 and contaminating CD31-negative cells.

Incubate with a dissociation reagent to detach the cells and filter to obtain a single-cell suspension.

Add a media to neutralize the enzymes, centrifuge to discard the supernatant, and resuspend the cells in a buffer.

Incubate with a blocking reagent to inhibit the cellular Fc receptors, preventing non-specific labeling.

Add a fluorophore-conjugated antibody specific for CD31 and incubate to label the EPCs.

Centrifuge and discard unbound antibodies. Incubate with antibody complexes containing one antibody that binds to the fluorophores.

Introduce polymer-coated magnetic nanoparticles and incubate, allowing the nanoparticles to bind to the complexes' second antibody.

Using a magnetic field, isolate the nanoparticle-bound cells.

Discard the suspension with unlabeled CD31-negative cells and resuspend the purified cells in a medium. The CD31-positive EPCs are ready for downstream assays.

On day 5 of the endothelial progenitor cell culturing, aspirate the medium from the wells and add 1 milliliter of dissociation reagent to each well. Incubate the plate for 6 to 8 minutes at 37 degrees Celsius. Using a micropipette, dissociate and singularize the cells, then, pass the cell suspension through a 40-micrometer cell strainer to filter into a 50-milliliter tube containing 10 milliliters of DMEM/F-12 medium.

To stop the digestion reaction, add DMEM/F-12/10 10 medium up to the 50-milliliter mark. Thoroughly pipette and reserve 10 microliters for counting the cells. Centrifuge the 50-milliliter tube at 200 g for 5 minutes at 20 to 25 degrees Celsius to pellet the cells. After centrifugation, remove the supernatant and resuspend the cell pellet with 10 milliliters of DMEM/F-12/10 medium.

Transfer the cell suspension into a fresh 15-milliliter tube. Centrifuge at 200 g for five minutes at 20 to 25 degrees Celsius to pellet the cells again. Then, resuspend the cell pellet into flow buffer 1. Next, add the FcR blocking reagent at a 1-to-100 ratio and incubate for 5 minutes. Then, add the 1-to-200 ratio diluted fluorescein isothiocyanate or FITC-labeled CD31 antibody.

Incubate the suspension for 30 minutes in the dark at a temperature between 20 and 25 degrees Celsius. At the end of the incubation, add 10 milliliters of flow buffer 1 to the sample, and reserve 10 microliters of the suspension for flow cytometry analysis to determine the fraction of CD31-positive cells. Centrifuge the cells at 200 g for 5 minutes at 20 to 25 degrees Celsius. After centrifugation, resuspend the cells with flow buffer 1 solution.

Next, add 5 microliters of the FITC selection cocktail per 100 microliters of the cell suspension. Mix the solution thoroughly by pipetting and incubate in the dark for 15 minutes at a temperature between 20 and 25 degrees Celsius. Next, add 5 microliters of magnetic nanoparticles per 100 microliters of cell suspension. Pipette the mixture well, and incubate in the dark for 10 minutes at 20 to 25 degrees Celsius.

Transfer the cell suspension to a 5-milliliter flow cytometry tube, and add flow buffer 1 to get a total volume of 2.5 milliliters. Then, place the flow cytometry tube in the magnet for five minutes. In a continuous motion, invert the magnet and decant the cell suspension containing FITC-CD31 antibody-unlabeled cells. Maintain the magnet and tube in the inverted position for 2 to 3 seconds, and then remove the remaining liquid.

Aspirate any droplets on the tube edge before returning the tube to an upright position. Retrieve the flow cytometry tube from the magnet, and add 2.5 milliliters of flow buffer 1 to wash the remaining CD31-positive cells. Gently pipette the cells up and down, two or three times to resuspend them. Then, place the flow tube into the magnet for five minutes.

Repeat the washing at least four times to remove the FITC-CD31 unlabeled cells from the tube. Remove the flow tube from the magnet, and resuspend the purified CD31-positive cells with the desired amount of a suitable medium. Reserve two 1-microliter aliquots of the suspension, one for cell counting, and the second to carry out flow cytometry analysis to assess the purity of CD31-positive cells in post-magnetic activated cell sorting samples.