Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

The overall goal of this experiment is to assess the immunomodulatory properties of human mesenchymal stem cells or msc. This method can help elucidate the mechanisms involved in human mesenchymal stem cell immunomodulation, a process that may have therapeutic relevance. The main advantage of this technique is that both the occurrence of effector, leukocyte proliferation and the number of cell divisions can be assessed demonstrating the procedure will be per shoe.

A technician from my lab To isolate pbmc begin by diluting 25 milliliters of a heparinized whole blood sample with 25 milliliters of PBS in a 50 milliliter conical tube. Next, add 15 milliliters of phi, call pack density gradient to a new 50 milliliter tube. Then holding the tube in an angle slowly layer the diluted cell suspension over the density gradient.

Taking care of that, there is no mixing of the layers. One of the most critical steps in this experiment is a layering of the blood without disturbing the density gradient. This is to ensure a high purity and low red blood cell contamination of the isolated pbmc.

Now, separate the cells by centrifugation. Three distinct layers should be apparent. The upper plasma layer, the bottom fial pack density gradient layer, and a thin middle PBMC layer.

Using a PE pipette, remove the upper layer taking care not to aspirate the interphase layer. Then use a 10 milliliter pipette to carefully transfer the pbmc to a 50 milliliter conical tube. Mix the cells with 20 milliliters of PBS and then spin them down to remove any traces of density gradient.

Then wash the pellet in another 20 milliliters of PBS to remove the platelets. Resus suspending the second pellet in complete leukocyte medium at a one times 10 of the seventh cells per milliliter concentration. To set up an MSC leukocyte co-culture prewarm, complete MSC medium to 37 degrees Celsius for no more than 30 minutes.

Then seed five times 10 to the fourth MSC per one milliliter of complete MSC medium per well in 24 well plates for overnight attachment at 37 degrees Celsius, allowing the cells to reach 80%confluence. The next morning, aspirate the medium and add the appropriate number of CFSE labeled P BMCs to the MSC in one milliliter of complete leukocyte medium. At a one to 10 MSC to PBMC ratio, activate the cells with 10 micrograms of PHA per milliliter of complete leukocyte medium.

Then on days three and five of co-culture transfer the appropriate number of co-culture to round bottom fax tubes and assess the proliferation of the CFSE labeled leukocytes by flow cytometric analysis to perform an effector suppression assay. Begin by setting up an M-S-C-P-B-M-C co-culture as just demonstrated with 2.5 times 10 to the sixth p BMCs co-culture with 250, 000 MSCs. To ensure enough MSC co-culture p BMCs for subpopulation selection.

After 48 to 72 hours at 37 degrees Celsius, select the MSC induced immunomodulatory leukocytes of interest with the appropriate magnetic beads. Then add CFSE labeled allogeneic CD four positive T cells in one milliliter of complete leukocyte medium per well at various experimental ratios. Next, add anti CD 3 28 conjugated microbeads at a one-to-one B to cell ratio to stimulate the CD four positive T-cell.

On the third day of co culture, assess the proliferation of the CFSE labeled CD four positive T cells by flow cytometry MSCs are adherent cells with a fibroblastic spindle shaped morphology. And pbmc and leukocytes are small, round non-adherent cells, which allows the clear distinction of these two cell populations. In a co-culture, when there is no proliferation, CFSE labeled cells are observed as one highly positive and sharply fluorescent peak when the cells have been activated.

Proliferation manifests as multiple smaller peaks with a loss and left shift of the fluorescence intensity when proliferation suppression has occurred. For example, in an MSC co-culture, the multiple smaller peaks decrease in number with a concomitant increase in fluorescence intensity as evidenced by the shifting of the peaks to the right and increases in the sharpness of the rightmost peak, which represents the non dividing cells in an effector suppression assay. The suppressive effect of the MSC on CD four positive T-cell proliferation can be further assessed in a dose dependent manner.

After watching this video, you should have a good of how to assess human mesenchymal stem cell immunomodulation. Don't forget that working with human cells can be hazardous and that precautions should always be taken.