Macrophage bioassay standardization to assess the anti-inflammatory activity of mesenchymal stromal cell-derived small extracellular vesicles

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-1 beta, 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 x 10(4) cells/well and 4 x 10(4) 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 x 10(4) 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. (c) 2022 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.