(682d) Three-Dimensional Dynamic Culture Improves Extracellular Vesicle Production and Cargo Profile of Human Mesenchymal Stem Cells through Altered Biogenesis

Human mesenchymal stem cells (hMSC) have been shown to exhibit therapeutic potentials in the context of tissue regeneration and immunomodulation. Various clinical trials utilize hMSC as cell therapies in disease models such as stroke, multiple sclerosis, cardiovascular disease and graft-versus-host disease. Studies have shown that the clinical efficacy of hMSC is mainly attributed to the paracrine effects via the hMSC secretome. As part of secretome, extracellular vesicles (EV) have been shown to encapsulate proteins, enzymes and metabolites that contribute to the therapeutic potential of hMSC for various diseases. hMSC-derived EV (hMSC-EV), including both microvesicles and exosomes, exert homing/migratory and paracrine effects to facilitate cell-cell communication and immune response in damaged tissue. Although the mechanisms by which EV mediate tissue regeneration and restore homeostasis are still under investigation (with several clinical trials ongoing), hMSC-EV hold potential as a cell-free therapeutic strategy in regenerative medicine with the high feasibility of biomanufacturing. However, similar to hMSC expansion, scale-up production of hMSC-EV has become a major challenge for clinical research in cell-free therapy. Long-term culture of hMSC results in loss of stem cell characteristics, which may compromise the quality, quantity and payload of EV. Studies have shown low efficiency of EV secretion under conventional monolayer culture of hMSC and altered cargo profiles in EV derived from senescent cells. Inconsistent EV properties with regard to the cargo and functional differences also have been reported. Thus, optimization of large scale production of hMSC-EV with effective therapeutic cargos is critical.

In current study, dynamic three-dimensional (3D) aggregation is applied to hMSC to improve the EV production by biogenesis. hMSC were cultured as 3D aggregates under wave motion to facilitate aggregation and EV secretion. A modified isolation strategy also was utilized to enrich EV efficiently. Compared to monolayer culture, hMSC-EV quantity was improved significantly under 3D dynamic culture. Moreover, the derived hMSC-EV from 3D culture exhibited smaller sizes compared to monolayer culture, indicating higher enrichment of the exosome population among hMSC-EV. Analysis of EV biogenesis markers from 3D hMSC aggregates demonstrated increased activation of the endosomal sorting complexes required for transport (ESCRT) pathway, which may contribute to the altered biogenesis. For functional evaluation, EV from 3D hMSC aggregates enhanced the stress resistance of adult stem cells and rejuvenated aged stem cells expressing cellular senescence after EV application to those cultures. Moreover, 2D and 3D hMSC-EV were shown to modulate the immune response in different patterns determined by T lymphocyte and macrophage phenotype assays. MicroRNA sequencing of EV revealed in detail the cargo profile differences for EV derived from 3D dynamic culture versus monolayer culture, which may explain some of the functional differences. In summary, this study provides a promising strategy for high quality EV production from hMSC with enhanced therapeutic potentials compared to conventional monolayer culture.

This work is in part supported by NIH (R01 NS102395) and NSF (#1743426).