Jun Yong Kim^1,2,3† , Won‑Kyu Rhim^1† , Seung‑Gyu Cha¹, Jiwon Woo¹, Joo Youn Lee⁴, Chun Gwon Park^2,3 and Dong Keun Han^1*

¹Department of Biomedical Science, CHA University, 335 Pangyo‑ro, Bun‑dang‑gu, Seongnam‑si, Gyeonggi‑do 13488, Republic of Korea. 
²Department of Biomedical Engineering, Sungkyunkwan University (SKKU), 2066 Seobu‑ro, Jangan‑gu, Suwon‑si, Gyeonggi‑do 16419, Republic of Korea.
³Intelligent Precision of Healthcare Convergence, SKKU Institute for Convergence, Sung‑kyunkwan University (SKKU), 2066 Seobu‑ro, Jangan‑gu, Suwon‑si, Gyeo‑nggi‑do 16419, Republic of Korea. 
⁴Xcell Therapeutics, 333, Yeongdong‑daero, Gangnam‑gu, Seoul 06188, Republic of Korea.

^†Jun Yong Kim and Won-Kyu Rhim contributed equally to this work
^*Correspondence: Dong Keun Han

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.