Isolation and Validation of Podoplanin-positive Mesenchymal Stem Cell Subpopulations Using an Indirect Magnetic Bead Strategy

Our research focused on isolating specific mesenchymal stem cell subset for cost-effective magnetic bead softening with high purity and readability. This protocol can be applied to any membrane protein using standard PE conjugated antibodies. To begin, gather the required reagents.

Select mesenchymal stem cells, or MSCs, from passages three to five that have reached 80 to 90%confluence. Discard the culture medium. Wash the cells twice with 10 milliliters of pre-warmed PBS.

Gently rock the vessel and discard the PBS. Add two milliliters of 0.25%trypsin-EDTA and incubate at 37 degrees Celsius for three to five minutes. Under a microscope, observe the cells until they round up and begin to detach.

Add four milliliters of culture medium containing 16.5%FBS to neutralize the trypsin. Gently pipette the cells to ensure complete detachment and transfer the cell suspension to a 15-milliliter centrifuge tube. Centrifuge the cell suspension at 300 g for three minutes and discard the supernatant.

Re-suspend the cell pellet in one milliliter of buffer. Count the cells to determine the total cell number. Centrifuge and discard the supernatant as previously demonstrated.

Re-suspend the cells with buffer to achieve a density of 100 million cells per milliliter. Transfer the cell suspension to a five milliliter polystyrene round-bottom tube. Add 10 microliters of Fc gamma RII blocker per 10 million cells.

Mix gently and incubate at room temperature for 10 minutes. Add five microliters of phycoerythrin conjugated, or PE conjugated, antihuman podoplanin, or PDPN antibody, per 10 million cells. Mix gently and incubate at room temperature for 15 to 20 minutes protected from light.

Add 10 microliters of selection cocktail per 10 million cells. Mix gently and incubate at room temperature for 15 minutes. Use a vortex mixer to mix the magnetic beads vial vigorously for 30 seconds.

Add five microliters of magnetic beads per 10 million cells. Mix gently and incubate at room temperature for 10 minutes. Place the five milliliter polystyrene round-bottom tube containing the labeled cell sample in the magnetic separation stand.

Add buffer to the tube to bring the total volume to 2.5 to three milliliters. And incubate at room temperature for five minutes. Holding the magnet and tube together as one unit, invert them to pour the supernatant into a new collection tube.

Place the collected PDPN negative cell fraction on ice. Repeat the magnetic separation procedure three times as previously demonstrated, pouring the supernatant into the same PDPN negative fraction collection tube after each separation. After the final wash, remove the tube containing the magnetically bound PDPN positive cell fraction from the magnetic separation stand.

Add three milliliters of buffer to re-suspend the positive cells. Transfer the positive fraction cell suspension to a fresh-labeled tube. Count the cells from both the positive and negative fraction tubes.

Record the cell yield for each fraction. Centrifuge the positive fraction cell suspension at 300 g for three minutes and discard the supernatant. Re-suspend the cell pellet in MEM alpha culture medium containing 16.5%FBS.

Adjust the cell density as needed and seed the cells into culture plates or flasks. Incubate the cells at 37 degrees Celsius in a 5%carbon dioxide incubator. After counting the cells, seed them onto a 100-millimeter culture dish at a density of three million cells per dish in 12 milliliters of culture medium.

24 hours after seeding, observe cell attachment. Replace the culture medium with fresh medium to remove residual magnetic beads and non-adherent cells. Harvest the cells by trypsinization.

Gently pipette the cell suspension up and down 10 to 15 times to dislodge and remove magnetic beads and residual antibodies. Assess the percentage of PDPN PE-positive cells by flow cytometry. Collect cells during the logarithmic growth phase.

Seed the cells into a 96-well plate. At different time points, add Cell Counting Kit-8m or CCK-8, reagent and measure absorbance according to the manufacturer's instructions. Tabulate the pre-sort and post-sort data for different time points in GraphPad Prism software, then click on data to plot the proliferation curve.

Following FC receptor blocking, antibody labeling, and magnetic separation, the final cell count was approximately 3.5 million cells for the PDPN positive fraction and 6.4 million cells for the PDPN negative fraction. Before magnetic sorting, the frequency of PDPN positive cells in the starting cell population was 38.7%After sorting, the positive fraction contained 97.9%PDPN PE-positive cells. Flow cytometry analysis of the negative fraction revealed that 4.30%of PDPN positive cells remained, indicating efficient depletion of PDPN positive cells from the negative fraction.

The proliferation curve of PDPN positive cells over three consecutive days of culture showed no significant difference compared to unsorted MSCs, indicating that the sorting strategy did not cause a detectable reduction in CCK-8-based proliferation or metabolic activity compared to unsorted MSCs. This approach enable research to study subpopulation with high purity and readability. The most important consideration in this protocol is gentle to maintain cell readability and remove unbound beads.

Follow this procedure, research can perform proliferation assays, follow sedimentary and deflation studies.