Bone marrow mesenchymal stromal cells in a 3D system produce higher concentration of extracellular vesicles (EVs) with increased complexity and enhanced neuronal growth properties

Abstract

Purpose Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have been demonstrated to possess great potential in preclinical models. An efficient biomanufacturing platform is necessary for scale up production for clinical therapeutic applications. The aim of this study is to investigate the potential differences in neuro-regenerative properties of MSC-derived EVs generated in 2D versus 3D culture systems. Method Human bone marrow MSCs (BM-MSCs) were cultured in 2D monolayer and 3D bioreactor systems. EVs were isolated using ultracentrifugation followed by size and concentration measurements utilizing dynamic light scattering (NanoSight) and by fluorescence staining (ExoView). Mouse trigeminal ganglia (TG) neurons were isolated from BALB/c mice and cultured in the presence or absence of EVs derived from 2D or 3D culture systems. Neuronal growth and morphology were monitored over 5 days followed by immunostaining for β3 tubulin. Confocal images were analyzed by Neurolucida software to obtain the density and length of the neurites. Results The NanoSight tracking analysis revealed a remarkable increase (24-fold change) in the concentration of EVs obtained from the 3D versus 2D culture condition. ExoView analysis showed a significantly higher concentration of CD63, CD81, and CD9 markers in the EVs derived from 3D versus 2D conditions. Furthermore, a notable shift toward a more heterogeneous phenotype was observed in the 3D-derived EVs compared to those from 2D culture systems. EVs derived from both culture conditions remarkably induced neurite growth and elongation after 5 days in culture compared to untreated control. Neurolucida analysis of the immunostaining images (β3 tubulin) showed a significant increase in neurite length in TG neurons treated with 3D- versus 2D-derived EVs (3301.5 μm vs. 1860.5 μm, P < 0.05). Finally, Sholl analysis demonstrated a significant increase in complexity of the neuronal growth in neurons treated with 3D- versus 2D-derived EVs (P < 0.05). Conclusion This study highlights considerable differences in EVs obtained from different culture microenvironments, which could have implications for their therapeutic effects and potency. The 3D culture system seems to provide a preferred environment that modulates the paracrine function of the cells and the release of a higher number of EVs with enhanced biophysical properties and functions in the context of neurite elongation and growth.

Oncostatin M-Enriched Small Extracellular Vesicles Derived from Mesenchymal Stem Cells Prevent Isoproterenol-Induced Fibrosis and Enhance Angiogenesis

“…Indeed, the ability of SEVs to interact with fibroblast receptors was previously described in the context of exosomes derived from cancer cell lines that expressed TGF-β. In this study, the capability of nanovesicles to induce fibroblast activation and myofibroblast differentiation in vitro was shown [ 51 , 52 ]. The expression of GP130 has also been reported to be triggered in the myocardial tissue of rats subjected to acute myocardial infarction as soon as one day after coronary artery ligation and was maintained for at least two months post-injury [ 53 ], which also suggests that SEVs containing OSM on their surface could interact with the afore mentioned receptors in vivo.…”

Production and Utility of Extracellular Vesicles with 3D Culture Methods

“…Additionally, a notable shift toward a more heterogenous phenotype was observed in microcarrier-based 3D-culture-derived EVs when compared to 2D-culture-derived EVs. Both 2D and microcarrier-based 3D-culture-derived EVs induced neurite growth, but microcarrier-based 3D-culture-derived EVs showed a significant increase in neurite length in trigeminal ganglia (TG) neurons when compared to 2D EVs in vitro [ 215 ].…”

Biomaterial-enabled 3D cell culture technologies for extracellular vesicle manufacturing

“…3D culture models have been used to induce EV production at an increased rate of up to 40-fold compared to 2D culture models. 72 A driving force for enhanced EV secretion in 3D culture models is the biomimetic extracellular structure which are found lacking in 2D models. These structures enable cell–cell interactions and cell–matrix interactions which can activate EV production.…”

“…89,90 Most importantly, 3D cell cultures have been shown to improve the yield and therapeutic quality of produced EVs. 70,72,90–101 3D cell cultures for EV production can be classified based on their 3D structure and culture conditions. These categories include adherent and non-adherent cultures that are maintained in static conditions, as well as dynamic conditions that are achieved through the use of perfusion reactors and microcarrier-based spinner bioreactors (Table 1).…”

“…For instance, human mesenchymal stem cells (hMSCs) are the preferred choice for large-scale production of therapeutic EVs due to their ability to produce high rates of EVs without altering their therapeutic potential. 28,70–72 Similarly, immune cells such as macrophages and dendritic cells also produce therapeutic EVs that can be utilized in immunotherapies by regulating inflammation and immune response. 73–75 However, EVs derived from cancer cells can promote tumour migration and survival by facilitating communication between tumour cells and tumour-associated fibroblasts.…”