Functional analysis of extracellular vesicles derived from human mesenchymal stromal cells used in cell therapy

Mesenchymal stromal cells (MSC) are heterogeneous cells capable of controlling the immune response, acting as an important tool in cell therapy, especially in the treatment of inflammatory and autoimmune diseases. Evidence suggests that extracellular vesicles derived from mesenchymal stromal cells (EV-MSC) has similar functions to MSC, transporting bioactive molecules to target cells, which can alter their phenotype or functional behavior. The objective of this work was the morphological and functional characterization of extracellular vesicles derived from MSC (EV-MSC) from different sources, cultured in a medium free of xenoantigens, under hypoxia and normoxia. For this purpose, an efficient protocol for depleting the extracellular vesicles of human AB serum was standardized and will be used in the production of EV-MSC. Then, the effect of depleted EV medium on the morphological, immunophenotypic, proliferation, viability and differentiation properties of MSC was evaluated. The results demonstrated that it is possible to isolate and characterize MSC from umbilical cord (UC-MSC) and adipose tissue (AT-MSC), in normoxic and hypoxic conditions, under conditions free of xenoantigens and depleted of EV, representing a major advance for production of EV-MSC. In addition, EV were isolated from the supernatant of the MSC by ultracentrifugation and characterized in terms of size and concentration, as well as protein expression. The expression of genes involved in immunoregulation present in EVs and their potential for inhibition and interaction with T lymphocytes was also evaluated. In conclusion, this work demonstrates that MSC-derived EVs have an immunomodulatory effect in conditions free of xenoantigens and pre-conditioned by hypoxia, with potential therapeutic application. (AU)