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Human mesenchymal stem cells (hMSCs)-derived extracellular vesicles (EVs) have been known to possess the features of the origin cell with nano size and have shown therapeutic potentials for regenerative medicine in recent studies as alternatives for cell-based therapies. However, extremely low production yield, unknown effects derived from serum impurities, and relatively low bioactivities on doses must be overcome for translational applications. As several reports have demonstrated the tunability of secretion and bioactivities of EVs, herein, we introduced three-dimensional (3D) culture and cell priming approaches for MSCs in serum-free chemically defined media to exclude side effects from serum-derived impurities. Aggregates (spheroids) with 3D culture dramatically enhanced secretion of EVs about 6.7 times more than cells with two-dimensional (2D) culture, and altered surface compositions. Further modulation with cell priming with the combination of TNF-α and IFN-γ (TI) facilitated the production of EVs about 1.4 times more than cells without priming (9.4 times more than cells with 2D culture without priming), and bioactivities of EVs related to tissue regenerations. Interestingly, unlike changing 2D to 3D culture, TI priming altered internal cytokines of MSC-derived EVs. Through simulating characteristics of EVs with bioinformatics analysis, the regeneration-relative properties such as angiogenesis, wound healing, anti-inflammation, anti-apoptosis, and anti-fibrosis, for three different types of EVs were comparatively analyzed using cell-based assays. The present study demonstrated that a combinatory strategy, 3D cultures and priming MSCs in chemically defined media, provided the optimum environments to maximize secretion and regeneration-related bioactivities of MSC-derived EVs without impurities for future translational applications. [ABSTRACT FROM AUTHOR]

Three-dimensional visualization of cellular and subcellular-structures in histological-tissues is essential for understanding the complexities of biological-phenomena, especially with regards structural and spatial relationships and pathologlical-diagnosis. Recent advancements in tissue-clearing technology, such as Magnified Analysis of Proteome (MAP), have significantly improved our ability to study biological-structures in three-dimensional space; however, their wide applicability to a variety of tissues is limited by long incubation-times and a need for advanced imaging-systems that are not readily available in most-laboratories. Here, we present optimized MAP-based method for paper-thin samples, Paper-MAP, which allow for rapid clearing and subsequent imaging of three-dimensional sections derived from various tissues using conventional confocal-microscopy. Paper-MAP successfully clear tissues within 1-day, compared to the original-MAP, without significant differences in achieved optical-transparency. As a proof-of-concept, we investigated the vasculature and neuronal-networks of a variety of human and rodent tissues processed via Paper-MAP, in both healthy and diseased contexts, including Alzheimer's disease and glioma. [ABSTRACT FROM AUTHOR]