Harnessing the mechanical microenvironment to optimize mesenchymal stem/stromal cells (MSCs) extracellular vesicle therapeutics

The advent of cell-free therapies has revolutionized regenerative medicine, positioning extracellular vesicles (EVs) as promising alternatives to direct stem cell transplantation. Among the diverse stem cell sources, mesenchymal stem/stromal cells (MSCs) have emerged as a leading candidate for EV production, owing to their relative ease of isolation, robust immunomodulatory capabilities, and broad tissue repair potential (1-7). Despite these advantages, the full therapeutic utility of MSC-derived EVs (MSC-EVs) remains contingent on optimizing the cellular microenvironment during culture. In particular, the mechanical cues within three-dimensional (3D) culture systems critically influence MSC behavior and the quality and function of their secreted EVs. In recent years, this concept has gained growing attention, as demonstrated by an increasing number of studies and clinical trials exploring mechanically conditioned MSC-EVs for applications ranging from musculoskeletal repair to cardiovascular and neurological regeneration.

In this context, the study by Kang et al. (8) offers timely and compelling insight into the critical role of mechanical cues in shaping the therapeutic potential of EVs derived from stem cells cultured in 3D environments. The authors engineer a series of tunable 3D hydrogel systems with varying mechanical properties to culture stem cells and assess the resultant EVs in terms of molecular cargo and functional activity. Their findings showed that subtle differences in matrix mechanical stiffness and viscoelasticity lead to significant alterations in the angiogenic and immunomodulatory functions of the released EVs. These results reinforce a key concept increasingly recognized in the field of regenerative medicine—that mechanotransduction, the cellular process of converting mechanical signals into biochemical activity, is not only a determinant of stem cell fate (9-11), but also a modulator of the paracrine factors they secrete, including EVs. By adopting a rigorous experimental design and a multidimensional analytical approach, Kang et al. also provide a valuable framework for future investigations aiming to dissect the link between mechanical conditioning and EV functionality, a critical step toward the rational design of EV-based therapeutics.

MSCs are inherently mechanosensitive cells that respond dynamically to their physical surroundings. In vivo, MSCs reside in mechanically heterogeneous niches such as bone marrow, adipose tissue, and umbilical cord, where the stiffness, viscoelasticity, and mechanical forces intricately regulate their fate, secretory profile, and paracrine signaling (10). Conventional two-dimensional culture systems inadequately recapitulate these complex biomechanical environments, often resulting in altered MSC behavior and suboptimal EV bioactivity. In contrast, 3D biomimetic culture platforms more faithfully reproduce the native extracellular matrix (ECM) mechanics and topology, enhancing MSC physiological relevance. Studies have demonstrated that tuning parameters such as matrix elasticity, porosity, and degradability significantly modulate MSC differentiation, secretory function, and importantly, the quantity and molecular composition of MSC-EVs (3,8). These findings underscore the concept that mechanical conditioning within 3D cultures can be strategically leveraged to “program” MSCs toward generating EVs enriched with specific therapeutic cargo, underscoring that engineering the cell culture microenvironment can be as influential as genetic or pharmacological modulation in directing the therapeutic profile of EVs.

However, Kang et al.’s work also prompts important questions. How durable are the functional enhancements imparted by mechanical conditioning? Do they persist in vivo under complex physiological conditions? How do different types of mechanical stimuli, beyond stiffness, such as shear stress, strain, or viscoelastic remodeling, affect EV cargo loading and release? Moreover, are there trade-offs in EV functionality or safety associated with such preconditioning?

At the molecular level, EV biogenesis and cargo loading are closely tied to mechanotransduction pathways and cytoskeletal dynamics. A more mechanistic perspective reveals that several canonical pathways link mechanical sensing to EV biogenesis. In particular, the yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ)-focal adhesion kinase (FAK) axis and integrin-cytoskeleton crosstalk act as central transducers of mechanical cues, regulating cytoskeletal tension, membrane dynamics, and vesicle trafficking. The YAP/TAZ-FAK axis is one of the most prominent, linking substrate stiffness and cytoskeletal tension to altered EV secretion and cargo composition through actin remodeling and transcriptional control of vesicle biogenesis genes. Similarly, integrin-cytoskeleton crosstalk regulates endosomal trafficking and membrane budding dynamics, thereby modulating EV release and surface marker profiles.

Together, these pathways couple extracellular mechanical stimuli to intracellular vesicle formation machinery, such as the endosomal sorting complexes required for transport complex (12) and Rab GTPases (13), providing a mechanistic framework for understanding and engineering EV output under defined biophysical conditions. For example, culturing MSCs within stiff 3D hydrogels, including polyethylene glycol- or gelatin methacrylate-based matrices, has been shown to activate YAP/TAZ signaling and integrin-mediated FAK pathways, culminating in enhanced EV secretion (14). This mechanical stimulation also enriches EV cargo with pro-angiogenic factors such as Vascular endothelial growth factor and miR-126, as well as anti-inflammatory mediators including miR-146a and tumor necrosis factor-stimulated gene (TSG)-6, which collectively enhance therapeutic outcomes in models of myocardial infarction, osteoarthritis, and neural injury (4,6,15-18). Conversely, softer matrices or dynamic mechanical stimuli such as shear stress and cyclic strain can bias EV contents toward neuroprotective or immunosuppressive profiles, suggesting the possibility of tailoring MSC-EVs for specific clinical indications through mechanical preconditioning (19-21). Moreover, MSC-EVs derived from mechanically optimized cultures exhibit improved stability, targeting efficiency via enhanced surface marker expression, and superior in vivo functional potency, further supporting the translational relevance of such approaches (22,23). For example, dynamic expansion of MSCs in stirred tank bioreactors significantly increases EV yield and enhances epitope marker expression, while maintaining particle integrity and in vitro functional potency (23). Similarly, MSCs cultured in perfusion bioreactors incorporating 3D-printed scaffolds produce EVs with preserved morphology and surface markers (e.g., CD63, Alix, TSG101), alongside superior pro-angiogenic bioactivity compared to static conditions (24). These findings collectively reinforce the notion that mechanical conditioning is not merely a passive culture parameter but a powerful bioengineering lever to enhance the potency, specificity, and reproducibility of EV-based therapeutics.

A major hurdle in clinical translation is the scalable production of potent MSC-EVs under good manufacturing practice (GMP) conditions. Mechanically engineered 3D bioreactor systems—such as spinner flasks, perfusion bioreactors, and dynamic hydrogel platforms—offer promising solutions by mimicking physiological mechanical loading while sustaining MSC stemness and secretory activity over extended culture periods (23-28). These systems facilitate increased EV yield without compromising quality and provide opportunities for standardization and reproducibility, which are critical for regulatory approval of EV-based therapeutics. Additionally, preconditioning MSCs with mechanical stimuli reflective of musculoskeletal or vascular environments has been shown to generate EVs with enhanced efficacy in bone regeneration, cartilage repair, and vascular remodeling. For example, MSCs subjected to dynamic compressive loading in chondrogenic hydrogels produced constructs with superior in vivo cartilage regeneration in osteochondral defect models (29). More directly, EVs derived from mechanically loaded MSCs have demonstrated enhanced chondrocyte targeting, ECM preservation, and therapeutic outcomes in osteoarthritis models (30). In the context of bone repair, several preconditioning approaches—including mechanical microenvironment modulation—have been shown to boost the regenerative efficacy of MSC-EVs in vivo, as reviewed systematically across multiple models (31). These results emphasize the potential of integrating mechanobiology with biomanufacturing to produce clinically relevant MSC-EVs, and suggest that establishing standardized mechanical preconditioning protocols within GMP-compliant workflows could greatly accelerate regulatory approval and clinical adoption, reducing batch-to-batch variability and ensuring consistent therapeutic efficacy. In this frame, translating mechanically conditioned EVs into GMP and clinical-grade products also presents significant regulatory challenges. Key issues include ensuring batch-to-batch consistency, defining potency assays that reflect mechanosensitive functional attributes, and establishing clear release criteria aligned with GMP standards. To support scalability and regulatory compliance, mechanical parameters, such as strain magnitude, frequency, or substrate stiffness, should be defined as critical process attributes within manufacturing protocols. Standardizing and validating these parameters would provide a measurable framework for process control, facilitating reproducibility and regulatory approval of mechanically optimized EV therapeutics.

In conclusion, it is increasingly clear that mechanical engineering of the 3D culture environment is central—not ancillary—to optimizing the therapeutic potential of MSC-EVs (Figure 1). The intersection of mechanobiology, stem cell engineering, and EV science represents a transformative platform for next-generation regenerative therapies. Future efforts should focus on systematically elucidating the relationships between mechanical parameters and EV cargo across MSC sources, designing smart biomaterials capable of dynamic mechanical tuning, integrating real-time EV analytics into bioreactors, and fostering collaborative standardization initiatives across academia and industry. Harnessing these mechanobiological principles will accelerate the development of potent, predictable, and programmable MSC-EVs tailored for a broad spectrum of clinical applications. The work by Kang et al. represents an important milestone in this rapidly evolving field, setting the stage for a new era of precision-engineered, mechanically optimized EV therapeutics. In parallel, the integration of digital monitoring and AI-driven bioreactor control holds promise for real-time EV quality management, enabling adaptive manufacturing and advancing the translational readiness of these next-generation biologics.

Figure 1 Mechanical engineering of the 3D culture environment is central to optimizing the therapeutic potential of MSC-EVs. EV, extracellular vesicle; MSC, mesenchymal stem/stromal cell; MSC-EVs, MSC-derived EVs.

Acknowledgments

None.

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, Annals of Translational Medicine. The article has undergone external peer review.

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