Related Experiment Video
Updated: Sep 5, 2025
Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions
Published on: March 18, 2017
Macrophage bioassay standardization to assess the anti-inflammatory activity of mesenchymal stromal cell-derived
Ricardo Malvicini1, Diego Santa-Cruz2, Giada De Lazzari3
1Laboratorio de Regulación Génica y Células Madre, Instituto de Medicina Traslacional, Trasplante y Bioingeniería, Universidad Favaloro-Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina; Department of Women's and Children's Health, University of Padua, Padua, Italy; Laboratory of Extracellular Vesicles as Therapeutic Tools, Fondazione Istituto di Ricerca Pediatrica Città della Speranza, Padua, Italy; LIFELAB Program, Consorzio per la Ricerca Sanitaria, Padua, Italy.
Background Aims:
Owing to the lack of biological assays, determining the biological activity of extracellular vesicles has proven difficult. Here the authors standardized an in vitro assay to assess the anti-inflammatory activity of mesenchymal stromal cell-derived small extracellular vesicles (MSC-sEVs) based on their ability to prevent acquisition of the M1 phenotype in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Induction of tumor necrosis factor alpha, IL-1β, IL-6 and inducible nitric oxide synthase (iNOS) characterizes the M1 phenotype. Nitric oxide released by iNOS turns into nitrite, which can be easily quantitated in culture media by Griess reaction.
Methods:
The authors first tested different assay conditions in 96-well plates, including two seeding densities (2 × 104 cells/well and 4 × 104 cells/well), four LPS doses (1 ng/mL, 10 ng/mL, 100 ng/mL and 1000 ng/mL) and two time points (16 h and 24 h), in order to determine the best set-up to accurately measure nitrite concentration as an index of M1 macrophage polarization.
Results:
The authors found that seeding 2 × 104 cells/well and stimulating with 10 ng/mL LPS for 16 h allowed the inhibition of nitrite production by 60% with the use of dexamethasone. Using these established conditions, the authors were able to test different MSC-sEV preparations and generate dose-response curves. Moreover, the authors fully analytically validated assay performance and fulfilled cross-validation against other M1 markers.
Conclusions:
The authors standardized a quick, cheap and reproducible in vitro macrophage assay that allows for the evaluation and estimation of the anti-inflammatory activity of MSC-sEVs.
