Bioassay standardization to assess exosomes antiinflammatory activity in vitro

MALVICINI, RICARDO; YANNARELLI, GUSTAVO GABRIEL; SANTA CRUZ, DIEGO MARIO; PACIENZIA, NATALIA ALEJANDRA

Rosario

Congreso; Reunión anual SAFIS 2019; 2019

Sociedad Argentina de Fisiología

Exosomes (Exo) are small sized-extracellular vesicles (40-150 nm), released by almost all kind of cells, and play amajor role in cell-to-cell communication. In the last years,they have drawn attention due to its potential applicationin clinical diagnostics and therapeutics. However,determining their biological activity and potency hasproven difficult, owing to the lack of biological assays.Here, we standardized an in vitro assay to assess the antiinflammatorypotential of mesenchymal stem cells(MSCs)-derived exosomes based on their ability to preventthe acquisition of the M1 phenotype in LPS-stimulatedRAW264.7 macrophages. M1 phenotype wascharacterized by the induction of IL-1β, IL-6 and iNOS asdetermined using qRT-PCR. Nitric oxide (NO) released byiNOS turns into NO2-, that can be easily quantitated in theculture media by Griess reaction. Moreover, phenol redpresent in culture media did not interfere in thespectrophotometric detection of NO2-. Thus, we firsttested 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 timepoints(16 and 24 h), in order to determine the best set-upto accurately measure NO2- production as a marker of M1macrophage polarization. We found that seeding 2x104cells/well and stimulating with 10 ng/ml LPS for 16 hallowed us to inhibit the inflammatory response by 60%using Dexamethasone (1 ug/ml). Using these establishedconditions, we were able to test different exosomespreparations in sextuplicate (5 μg protein/well) and torank then by their anti-inflammatory activity. Finally,conditioned media containing NO2- can be processedimmediately or stored at -20°C, as we found that NO2- isstable in culture media. In summary, we standardized aquick, cheap and reproducible in vitro macrophage assaythat allows evaluating and estimating the antiinflammatoryactivity of MSCs-derived exosomes. Theassay is convenient for comparing multiple samples and,therefore, should be useful in developing protocols toimprove the purification and characterization of antiinflammatoryexosomes