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 &lt; 0.05 vs. vehicle control) and dose-dependent stimulation of dermal papilla cell growth <i>in vitro</i>. Importantly, these cellular effects translated into therapeutic outcomes, with the EVs significantly promoting hair regeneration <i>in vivo</i>. This innovative manufacturing strategy holds great potential to accelerate the clinical translation of cell-free immune and regenerative therapies via leveraging hucMSC-EVs.