Development of a flow cytometry-based potency assay for measuring the in vitro immunomodulatory properties of mesenchymal stromal cells

• Potency assay for immunosuppressive properties of mesenchymal stromal cells.
• A rapid assay using flow cytometry of cultured whole blood.
• Importance of anticoagulant in blood collection.
• Calcium-dependence of monocyte activation by LPS.

Human bone marrow-derived mesenchymal stromal/stem cells (MSC) have well-documented modulatory effects on multiple immune cell types. Although these effects are linked to their therapeutic benefit in diverse diseases, a reliable, quantitative assay of the immunomodulatory potency of individual human MSC preparations is lacking. The aims of this study were to develop an optimised rapid turnaround, flow cytometry-based whole-blood assay to monitor MSC potency and to validate its application to MSC immunomodulation. A protocol for short-term LPS stimulation of anti-coagulated whole blood samples followed by combined surface CD45/CD14 and intracellular TNF-α staining was initially developed for analysis on a 4 colour desktop cytometer. Optimal monocyte activation was dependent on the presence of extracellular calcium ions thereby precluding the use of EDTA and sodium citrate as anticoagulants. Optimal assay conditions proved to be 1 ng/mL ultrapure-LPS added to 10-fold diluted, heparin anti-coagulated whole blood incubated for 6 h at 37 °C. Under these conditions, addition of human bone marrow-derived MSC (hBM-MSC) from multiple donors resulted in a reproducible, dose-dependent inhibition of LPS-stimulated monocyte TNF-α expression. We conclude that this protocol represents a practical, quantitative assay of a clinically relevant functional effect of hBM-MSCs as well as other immunomodulatory agents.