Bioassay standardization to assess exosomes antiinflammatory activity in vitro.

MALVICINI, RICARDO; SANTA CRUZ, D; PACIENZA, N; YANNARELLI G.

Congreso; REUNIÓN ANUAL SAFIS 2019; 2019

Exosomes (Exo) are small sized-extracellular vesicles (40-150 nm), released by almost all kind of cells, and play a major role in cell-to-cell communication. In the last years, they have drawn attention due to its potential application in clinical diagnostics and therapeutics. However, determining their biological activity and potency has proven difficult, owing to the lack of biological assays. Here, we standardized an in vitro assay to assess the anti-inflammatory potential of mesenchymal stem cells (MSCs)-derived exosomes based on their ability to prevent the acquisition of the M1 phenotype in LPS-stimulated RAW264.7 macrophages. M1 phenotype was characterized by the induction of IL-1β, IL-6 and iNOS as determined using qRT-PCR. Nitric oxide (NO) released by iNOS turns into NO2-, that can be easily quantitated in the culture media by Griess reaction. Moreover, phenol red present in culture media did not interfere in the spectrophotometric detection of NO2-. Thus, we first tested different assay conditions in 96-well-plates, including two seeding densities (2x104 and 4x104 cells), four LPS doses (1, 10, 100 and 1000 ng/ml) and two time-points (16 and 24 h), in order to determine the best set-up to accurately measure NO2- production as a marker of M1 macrophage polarization. We found that seeding 2x104 cells/well and stimulating with 10 ng/ml LPS for 16 h allowed us to inhibit the inflammatory response by 60% using Dexamethasone (1 ug/ml). Using these established conditions, we were able to test different exosomes preparations in sextuplicate (5 μg protein/well) and to rank then by their anti-inflammatory activity. Finally, conditioned media containing NO2- can be processed immediately or stored at -20°C, as we found that NO2- is stable in culture media. In summary, we standardized a quick, cheap and reproducible in vitro macrophage assay that allows evaluating and estimating the anti-inflammatory activity of MSCs-derived exosomes. The assay is convenient for comparing multiple samples and, therefore, should be useful in developing protocols to improve the purification and characterization of anti-inflammatory exosomes.