A novel approach for enumeration of extracellular vesicles from crude and purified cell culture samples

Extracellular Vesicles

Kruse, Thomas, Samuel Schneider, Lucas Nik Reger, Markus Kampmann, and Oscar‐Werner Reif. 2022. “A Novel Approach for Enumeration of Extracellular Vesicles from Crude and Purified Cell Culture Samples.” Engineering in Life Sciences, January. https://doi.org/10.1002/elsc.202100149.

The interest in extracellular vesicles (EVs) has been increased in recent years due to their potential application in diagnosis and therapy of severe diseases. The versatile fields of application due to the numerous possible cargos and the targeted delivery system make them a promising biopharmaceutical product. However, their broad size range as well as varied surface protein content result in challenges for the purification, characterization, and quantification. In this study a novel method, based on high-resolution flow cytometry, was examined for the enumeration of EVs in purified as well as crude process samples. In addition to quantification, samples were characterized by dynamic light scattering, zeta potential measurement, and analytical size exclusion chromatography. It has been demonstrated that EVs were successfully enumerated with the novel method, offering great benefits for development and monitoring of EV processes.

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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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