Bioassay standardization to assess exosomes antiinflammatory activity in vitro

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

paris

Congreso; International society for cell and gene therapy 2020; 2020

International society for cell and gene therapy

Background & Aim Exosomes (Exo) are small sized-extracellularvesicles (40- 150 nm), released by almost all kind of cells, and play amajor role in cell-to-cell communication. In the last years, they havedrawn attention due to its potential application in clinical diagnosticsand therapeutics. However, determining their biological activity andpotency has proven difficult, owing to the lack of biological assays.Here, we standardized an in vitro assay to assess the antiinflammatory potential of mesenchymal stem cells (MSCs)-derived exosomesbased on their ability to prevent the acquisition of the M1 phenotypein LPS-stimulated RAW264.7 macrophages.Methods, Results & Conclusion M1 phenotype was characterizedby the induction of IL-1b, IL-6 and iNOS as determined usingqRT-PCR. Nitric oxide (NO) released by iNOS turns into NO2-, thatcan be easily quantitated in the culture media by Griess reaction.Moreover, phenol red present in culture media did not interferein the spectrophotometric detection of NO2-. Thus, we first testeddifferent assay conditions in 96-well-plates, including two seeding densities (2 £ 104 and 4 £ 104 cells), four LPS doses (1, 10,100 and 1000 ng/ml) and two timepoints (16 and 24 h), in orderto determine the best set-up to accurately measure NO2- production as a marker of M1 macrophage polarization. We found thatseeding 2 £ 104 cells/well and stimulating with 10 ng/ml LPS for16 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 insextuplicate (5 mg protein/well) and to rank then by their antiinflammatory activity. Finally, conditioned media containing NO2-can be processed immediately or stored at -20°C, as we foundthat NO2- is stable in culture media.In summary, we standardized a quick, cheap and reproducible invitro macrophage assay that allows evaluating and estimating theantiinflammatory activity of MSCs-derived exosomes. The assay isconvenient for comparing multiple samples and, therefore, should beuseful in developing protocols to improve the purification and characterization of antiinflammatory exosomes.