In-vitro macrophage assay predicts the in-vivo anti-inflammatory potential of human mesenchymal stem cells-derived exosomes

SANTA-CRUZ D.; MALVICINI R.; YANNARELLI G.; PACIENZA N.

Mar del Plata

Congreso; LXIII Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica (SAIC); 2018

Sociedad Argentina de Investigación Clínica (SAIC)

Exosomes play key roles in cell biology and may provide new clinical diagnostics and therapies. However, one of the major barriers in the field has been a lack of convenient assays for their bioactivity. Here, we developed an in vitro assay that quantitates the anti-inflammatory potential of mesenchymal stem cells (MSC)-derived exosomes based on their ability to suppress the acquisition of M1 phenotype in LPS-stimulated RAW264.7 macrophages. We first standardized the LPS dose (10 ng/ml) that induces macrophage cells to secrete a moderate level of IL-6 which can be significantly suppressed by dexamethasone. To test the assay, we purified exosomes from seven independent lots of MSCs-conditioned medium by anion exchange chromatography. All the preparations were positive for human-specific CD63 and CD81, and showed a mode size ranging from 99 to 156 nm by nanoparticle tracking analysis. Dose-response curves indicated that a dose of 0.5x109 exosomes/ml provides reproducible results on categorizing the anti-inflammatory potential. To assess the predictive efficacy of the assay, the same seven exosome preparations were tested in a murine model of LPS-induced systemic inflammation. The inflammatory response was followed by expression of IL-1β and IL-6 in spleen at 2 h after LPS administration (2.5mg/Kg). The percentage of reduction in secreted IL-6 from the macrophage assay correlated not only with the suppression of mRNA levels for IL-1β (r2=0.812,p=0.006) and IL-6 (r2=0.861, p=0.003), but also with the reduction of IL-1β(r2=0.694, p=0.020) and IL-6 (r2=0.811, p=0.006) contents in spleen of mice receiving exosomes after LPS administration. In conclusion, the macrophage assay allows to rank different preparations of exosomes by their anti-inflammatory activity, and their ranking predicts their efficacy in suppressing inflammation in mice. The assay is convenient for comparing multiple samples and therefore should be useful in developing protocols for the purification and characterization of anti-inflammatory exosomes.