Human mesenchymal stem cell (hMSC) derived extracellular vesicles (EVs) have shown therapeutic potential in recent studies. However, the corresponding therapeutic components are largely unknown, and scale‐up production of hMSC EVs is a major challenge for translational applications. In the current study, hMSCs were grown as 3D aggregates under wave motion to promote EV secretion. Results demonstrate that 3D hMSC aggregates promote activation of the endosomal sorting complexes required for transport (ESCRT)‐dependent and ‐independent pathways. mRNA sequencing revealed global transcriptome alterations for 3D hMSC aggregates. Compared to 2D‐hMSC‐EVs, the quantity of 3D‐hMSC‐EVs was enhanced significantly (by 2‐fold), with smaller sizes, higher miR‐21 and miR‐22 expression, and an altered protein cargo (e.g., upregulation of cytokines and anti‐inflammatory factors) uncovered by proteomics analysis, possibly due to altered EV biogenesis. Functionally, 3D‐hMSC‐EVs rejuvenated senescent stem cells and exhibited enhanced immunomodulatory potentials. In summary, this study provides a promising strategy for scalable production of high‐quality EVs from hMSCs with enhanced therapeutic potential.

Human Mesenchymal Stem Cells (hMSCs) and their derived products hold potential in tissue engineering and as therapeutics in a wide range of diseases. hMSCs possess the ability to aggregate into “spheroids”, which has been used as a preconditioning technique to enhance their therapeutic potential by upregulating stemness, immunomodulatory capacity, and anti-inflammatory and pro-angiogenic secretome. Few studies have investigated the impact on hMSC aggregate properties stemming from dynamic and static aggregation techniques. hMSCs’ main mechanistic mode of action occur through their secretome, including extracellular vesicles (EVs)/exosomes, which contain therapeutically relevant proteins and nucleic acids. In this study, a 3D printed microchannel bioreactor was developed to dynamically form hMSC spheroids and promote hMSC condensation. In particular, the manner in which dynamic microenvironment conditions alter hMSC properties and EV biogenesis in relation to static cultures was assessed. Dynamic aggregation was found to promote autophagy activity, alter metabolism toward glycolysis, and promote exosome/EV production. This study advances our knowledge on a commonly used preconditioning technique that could be beneficial in wound healing, tissue regeneration, and autoimmune disorders.