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

Cytotherapy. 2022 Oct;24(10):999-1012. doi: 10.1016/j.jcyt.2022.05.011. Epub 2022 Jul 5.


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.

Keywords: bioactivity; extracellular vesicles; in-process assay; macrophage polarization; mesenchymal stromal cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Inflammatory Agents / pharmacology
  • Biological Assay
  • Extracellular Vesicles* / metabolism
  • Lipopolysaccharides / pharmacology
  • Macrophages / metabolism
  • Mesenchymal Stem Cells* / metabolism
  • Nitric Oxide Synthase Type II / metabolism
  • Nitrites / metabolism
  • Reference Standards


  • Anti-Inflammatory Agents
  • Lipopolysaccharides
  • Nitrites
  • Nitric Oxide Synthase Type II