title: Development of a flow cytometry-based potency assay for measuring the in vitro immunomodulatory properties of mesenchymal stromal cells.
abstract: 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 1ng/mL ultrapure-LPS added to 10-fold diluted, heparin anti-coagulated whole blood incubated for 6h 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.
authors.affiliation: Regenerative Medicine Institute, National Centre for Biomedical Engineering Science and School of Medicine, National University of Ireland, Galway, Ireland. Electronic address: andreia.ribeiro@nuigalway.ie.
authors.forename: Andreia
authors.initials: A
authors.lastname: Ribeiro
authors.affiliation: Regenerative Medicine Institute, National Centre for Biomedical Engineering Science and School of Medicine, National University of Ireland, Galway, Ireland. Electronic address: thomas.ritter@nuigalway.ie.
authors.forename: Thomas
authors.initials: T
authors.lastname: Ritter
authors.affiliation: Regenerative Medicine Institute, National Centre for Biomedical Engineering Science and School of Medicine, National University of Ireland, Galway, Ireland. Electronic address: matthew.griffin@nuigalway.ie.
authors.forename: Matthew
authors.initials: M
authors.lastname: Griffin
authors.affiliation: Regenerative Medicine Institute, National Centre for Biomedical Engineering Science and School of Medicine, National University of Ireland, Galway, Ireland. Electronic address: rhodri.ceredig@nuigalway.ie.
authors.forename: Rhodri
authors.initials: R
authors.lastname: Ceredig
mesh_terms: D000925:Anticoagulants / Q000378:metabolism; D001854:Bone Marrow Cells / Q000473:pathology; D002118:Calcium / Q000378:metabolism; D018929:Cell Culture Techniques; D002469:Cell Separation; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D056747:Immunomodulation; D017493:Leukocyte Common Antigens / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008070:Lipopolysaccharides / Q000276:immunology; D059630:Mesenchymal Stem Cells / Q000276:immunology* / Q000473:pathology; D009000:Monocytes / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D000925:Anticoagulants; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D017493:Leukocyte Common Antigens; D002118:Calcium
keywords: Flow cytometry; Immunomodulatory potency; Mesenchymal stromal cells; Monocytes; TNF-α; Whole blood assay