A high-throughput methodology for the efficient isolation of highly pure extracellular vesicles from skeletal muscle myoblasts

Extracellular Vesicles

Background: Skeletal muscle extracellular vesicles (SM-EVs) regulate gene expression events in myogenic differentiation. Optimising effective SM-EV isolation methods offering high levels of purity will be important to accurately define their composition and functionality. Size-exclusion chromatography (SEC) applied in combination with ultrafiltration (UF) has the potential to increase sample throughput, scalability and selectivity. However, an optimal UF+SEC methodology has not been tested for the isolation of myotube derived EVs. Our aim was to compare two different UF protocols and define an optimal window of SEC fractions to maximise SM-EVs recovery and sample purity. Methods: C2C12 myotube conditioned medium was pre-concentrated using Amicon® Ultra 15 or Vivaspin®20, 100KDa UF columns and processed by SEC (IZON, qEV 70nm). The resulting thirty fractions obtained were individually analysed to identify an optimal fraction window for EV recovery. Results: EV markers Alix and TSG101 could be detected up to fraction 13, while CD9 and Annexin A2 only up to fraction 6. ApoA1+ lipoprotein contaminants were detected from fraction 6 onwards for both protocols. Amicon and Vivaspin UF preconcentration protocols led to qualitative and quantitative variations in EV marker profiles and purity. Eliminating lipoprotein co-isolation by reducing the SEC fraction window resulted in a net loss of particles, but increased measures of sample purity and had only a negligible impact on the presence of EV marker proteins. Conclusion: In conclusion, this study developed optimal UF+SEC protocols for the isolation of SM-EVs based on sample purity (fractions 1-5) and total abundance (fractions 2-10). The resulting protocols will be valuable in isolating highly pure SM-EV preparations for biomarker studies.

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Cigarette smoke (CS) represents one of the most relevant environmental risk factors for several chronic pathologies. Tissue damage caused by CS exposure is mediated, at least in part, by oxidative stress induced by its toxic and pro-oxidant components. Evidence demonstrates that extracellular vesicles (EVs) released by various cell types exposed to CS extract (CSE) are characterized by altered biochemical cargo and gained pathological properties. In the present study, we evaluated the content of oxidized proteins and phospholipid fatty acid profiles of EVs released by human bronchial epithelial BEAS-2B cells treated with CSE. This specific molecular characterization has hitherto not been performed. After confirmation that CSE reduces viability of BEAS-2B cells and elevates intracellular ROS levels, in a dose-dependent manner, we demonstrated that 24 h exposure at 1% CSE, a concentration that only slight modifies cell viability but increases ROS levels, was able to increase carbonylated protein levels in cells and released EVs. The release of oxidatively modified proteins via EVs might represent a mechanism used by cells to remove toxic proteins in order to avoid their intracellular overloading. Moreover, 1% CSE induced only few changes in the fatty acid asset in BEAS-2B cell membrane phospholipids, whereas several rearrangements were observed in EVs released by CSE-treated cells. The impact of changes in acyl chain composition of CSE-EVs accounted for the increased saturation levels of phospholipids, a membrane parameter that might influence EV stability, uptake and, at least in part, EV-mediated biological effects. The present in vitro study adds new information concerning the biochemical composition of CSE-related EVs, useful to predict their biological effects on target cells. Furthermore, the information regarding the presence of oxidized proteins and the specific membrane features of CSE-related EVs can be useful to define the utilization of circulating EVs as marker for diagnosing of CS-induced lung damage and/or CS-related diseases.

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