INFLUENCE OF THE ISOLATION METHOD ON THE CHARACTERISTICS AND FUNCTIONAL ACTIVITY OF MESENCHYMAL STROMAL CELL-DERIVED EXTRACELLULAR VESICLES

MALVICINI, R; SANMARTIN, C; TOLOMEO, ANNA MARIA; SANTA CRUZ, DM; MURACA, MAURIZIO; PACIENZA, N; YANNARELLI G.

Congreso; LXVII REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA (SAIC); 2022; 2022

Extracellular vesicles (EVs) isolation methods are based on different physicochemical properties and may result in the purification of distinct EV populations.Objective: we compared two different isolation methods suitable for producing clinical-grade mesenchymal stromal cells-derived EVs, ion exchange chromatography (IEX) and ultrafiltration (UF), and we evaluated their impact on the composition and functional properties of the EVs.Methods: EVs were purified from the conditioned culture medium using an anion exchange resin (IEX) or Amicon filters (UF, 100kDa cut-off). We assessed nanoparticle size and distribution by NTA and tRPS, and morphology by TEM. We also measured protein, lipid, and total RNA concentration, and immunophenotyped both EV populations by flow cytometry (Macsplex assay). Moreover, immunomodulatory activity was tested using a standardized macrophage polarization assay and T cell stimulation assay.Results: We found that IEX and UF yielded a comparable amount of total particles (7.43E+10 vs. 7.04E+10) with similar size and distribution (mode 96nm vs. 97nm and 94nm vs. 101nm by tRPS and NTA, respectively). In addition, a similar amount of proteins (559±126µg vs. 479±83µg) and lipids (16±0.21µg vs. 18±0.29µg) was obtained with the two procedures. However, IEX yielded a 10-fold higher RNA quantity than UF (p