Protocol for Mesenchymal Stem Cell Immune Modulation
Mesenchymal stem cells (MSCs) have the unique potential to modulate the immune response by preventing the proliferation of peripheral blood mononuclear cells (PBMCs). Immune modulation function can be assayed in vitro by the co-culture of MSCs with pre-fluorescently labeled and activated PBMCs. By using flow cytometry analysis for the level of fluorescence intensity per cell after co-culture, this can serve as an indirect way to quantify the functional ability of MSCs to suppress immune activation.
- Mesenchymal stem cells
- Peripheral blood mononuclear cells (PBMCs)
- RPMI Medium 1640 (Irvine Scientific, Catalog #9161) containing 10% MSC qualified Fetal Bovine Serum
- CellTrace™ CFSE
- Phytohemagglutinin (PBMC stimulant) (Irvine Scientific, Catalog # 96691)
- Flow cytometer with 488nm excitation and emission filters
- Six-well plates
Mesenchymal Stem Cell Immune Modulation on T cells
1) Thaw and seed MSCs at 3,000 cells/cm2 in six-well plates. See Thawing Procedure.
2) Allow MSCs to reach 80% confluence before adding the PBMCs. MSCs should be approximately 500,000 cells per well before proceeding.
3) Pre-warm complete RPMI Medium 1640 containing 10% fetal bovine serum (MSC qualified) to 37°C for no more than 30 minutes.
4) Thaw PBMCs according to vendor’s instructions. Incubate cells at 37°C, 5% CO2 for 3 hours to recover.
5) After recovery, stain PBMCs with CellTrace™ CFSE according to manufacturer’s instructions.
6) After staining, wash the PBMCs three times using fresh, room temperature complete RPMI medium.
7) On the last wash, resuspend the PBMC cell pellet in minimal volume of complete RPMI medium.
8) Based on MSC cell counts, co-culture labeled PBMCs with MSCs in a six-well plate in a 1:10 ratio (MSC: PBMCs).
9) Add phytohemagglutinin to a final concentration of 20μg/mL in a total volume of 4mL complete RPMI per well.
10) Prepare a negative control sample by plating 250,000 PBMCs per well in a six-well plate with 4mL complete RPMI medium.
11) Prepare a positive control sample by plating 250,000 PBMCs per well in a six-well plate with 4mL complete RPMI medium supplemented with a final concentration of 20μg/mL phytohemagglutinin.
12) On day three and day five of co-culture, PBMC-derived CD3+ cell proliferation can be measured by using a flow cytometer with 488nm excitation and emission filters appropriate for fluorescein.
*CellTrace™ is a registered trademark of Life Technologies