Abstract
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.
Graphical Abstract
Graphical Abstract
Key Words
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Article info
Publication history
Published online: July 04, 2022
Accepted:
May 30,
2022
Received:
April 1,
2022
Identification
Copyright
© 2022 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.
