# Comprehensive Characterization and In Vitro Functionality Study of Small Extracellular Vesicles Isolated by Different Purification Methods from Mesenchymal Stem Cell Cultures

## TL;DR

This paper studies how different methods to purify tiny cell particles from stem cells affect their ability to heal tissues in lab tests.

## Contribution

The study compares purification methods for small extracellular vesicles from MSCs and links their purity to in vitro functionality.

## Key findings

- MSC-sEVs purified by SEC or DG-UC were characterized using advanced analytical methods.
- Purity levels of sEVs obtained by different methods correlate with their regenerative properties in vitro.
- Scratch wound healing and tube formation assays showed biological effects of purified sEVs.

## Abstract

Mesenchymal stem cells (MSCs) exhibit therapeutic properties, which have been attributed to their secretome, the set of secreted factors comprising cytokines, growth factors, and extracellular vesicles (EVs). In particular, small extracellular vesicles (sEVs) or exosomes, ranging between 30 nm and 120 nm in diameter, can target specific tissues to deliver molecular payloads, thus lending themselves as promising platform for cell-free therapies. In this study, sEVs were purified from the conditioned medium (CM) harvested from human bone marrow-derived MSC culture and purified using size-exclusion chromatography (SEC) or density gradient ultracentrifugation (DG-UC). Then sEVs were fully characterized for identity and integrity using multiple analytical methods, including single-particle, transcriptomic and proteomic analyses. Different in vitro cell-based assays were established to evaluate the biological effects of the purified sEVs. Specifically, scratch wound healing and tube formation assays using human umbilical vein endothelial cells (HUVECs) were used to evaluate the regenerative properties of MSC-sEVs. Our findings demonstrated that the in vitro functional properties of MSC-sEVs are correlated with sEVs’ purity levels obtained by different purification methods.