Unlocking the Therapeutic Potential of Mesenchymal Stromal Cell-Derived Extracellular Vesicles: A GMP-Compliant, Scalable Manufacturing Strategy

Human umbilical cord mesenchymal stromal cell-derived extracellular vesicles (hucMSC-EVs) emerge as promising nanotherapeutics for immunomodulation and tissue repair, yet their clinical translation has been constrained by critical manufacturing challenges in scalability, purity, and stability. Here, we developed a novel Good Manufacturing Practice-compliant platform combining serum-free/xeno-free three-dimensional microcarrier culture with downstream tangential flow filtration and multimodal anion exchange chromatography. The integrated process achieved ultra-pure hucMSC-EVs [(1.96 ± 0.11) × 10⁹ particles/μg protein; 99.81% protein clearance] at 5.59 × 10¹³ particles per 15-L batch—sufficient for Phase I/II trials (400-subject trials at 0.50–1.40 × 10¹¹ particles/dose). Notably, our lyophilized formulation maintained EV structural integrity and biological activity under thermal stress (40°C for 1 month) and accelerated storage conditions (25°C for 3 months), effectively addressing stability challenges. Functionally, the dual immunomodulatory and regenerative capacities of hucMSC-EVs were demonstrated through both significant suppression of phytohemagglutinin-induced peripheral blood mononuclear cell proliferation (50% inhibition, p < 0.05 vs. vehicle control) and dose-dependent stimulation of dermal papilla cell growth in vitro. Importantly, these cellular effects translated into therapeutic outcomes, with the EVs significantly promoting hair regeneration in vivo. This innovative manufacturing strategy holds great potential to accelerate the clinical translation of cell-free immune and regenerative therapies via leveraging hucMSC-EVs.