A scalable coaxial bioprinting technology for mesenchymal stem cell microfiber fabrication and high extracellular vesicle yield

Extracellular Vesicles

Chen, J., Zhou, D., Nie, Z., Lu, L., Lin, Z., Zhou, D., Zhang, Y., Long, X., Fan, S. and Xu, T., 2021. A scalable coaxial bioprinting technology for mesenchymal stem cell microfiber fabrication and high extracellular vesicle yield. Biofabrication, 14(1), p.015012.

Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) are promising candidates for regenerative medicine; however, the lack of scalable methods for high quantity EV production limits their application. In addition, signature EV-derived proteins shared in 3D environments and 2D surfaces, remain mostly unknown. Herein, we present a platform combining MSC microfiber culture with ultracentrifugation purification for high EV yield. Within this platform, a high quantity MSC solution (∼3 × 108 total cells) is encapsulated in a meter-long hollow hydrogel-microfiber via coaxial bioprinting technology. In this 3D core–shell microfiber environment, MSCs express higher levels of stemness markers (Oct4, Nanog, Sox2) than in 2D culture, and maintain their differentiation capacity. Moreover, this platform enriches particles by ∼1009-fold compared to conventional 2D culture, while preserving their pro-angiogenic properties. Liquid chromatography-mass spectrometry characterization results demonstrate that EVs derived from our platform and conventional 2D culturing have unique protein profiles with 3D-EVs having a greater variety of proteins (1023 vs 605), however, they also share certain proteins (536) and signature MSC-EV proteins (10). This platform, therefore, provides a new tool for EV production using microfibers in one culture dish, thereby reducing space, labor, time, and cost.

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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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