Engineering extracellular vesicles by three-dimensional dynamic culture of hMSC

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. Researchers at Florida State University grew hMSCs as 3D aggregates under wave motion to promote EV secretion. Results demonstrated 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.

Culture conditions for 2D and 3D hMSCs to promote EV secretion

(a) Illustration of 3D dynamic culture of hMSCs to form cell aggregates using a miniaturized wave bioreactor. (b) Schematic illustration of procedures from initial 2D expansion of hMSCs to the medium collection of 2D and 3D cultures and isolation of 2D-hMSC-EVs and 3D-hMSC-EVs. (c) Representative images of 3D hMSC aggregates and 2D culture of hMSCs. Scale bar: 200 μm (white), 50 μm (black). (d) Stem cell gene expression and (e) Colony forming unit-fibroblast (CFU-F) colony number from 2D or dynamic 3D culture of hMSCs in EV-depleted complete culture medium (CCM). n = 3. *P < 0.05; **P < 0.01; ***P < 0.001