Optimization of extrusion-based production of Wharton's jelly MSC-derived nanovesicles

Extracellular vesicles (EVs) have attracted increasing attention as therapeutic agents and drug-delivery nanocarriers. However, their clinical translation remains limited by low yield and scalability challenges. Recent studies demonstrated that extrusion-derived nanovesicles exhibit EV-like physicochemical properties and biological functions. Nevertheless, systematic optimization of extrusion-processing parameters for Wharton's jelly mesenchymal stem cell-derived extrusion nanovesicles (WJ-exNVs) remains insufficiently explored. Therefore, this study aimed to evaluate key extrusion-processing parameters influencing WJ-exNVs production.

Three extrusion-processing parameters, including extrusion cycle number, resuspension buffer, and input cell number, were systematically evaluated. WJ-exNVs were characterized using nanoparticle tracking analysis, transmission electron microscopy, bicinchoninic acid protein assay, and flow cytometry. Preliminary biological activity and short-term stability were also assessed.

Increasing extrusion cycles up to ten improved vesicle homogeneity and particle recovery, whereas additional extrusion did not further enhance productive vesicle formation. Phosphate-buffered saline showed slightly higher particle recovery than ddH₂O with limited physicochemical differences. The 2.5 × 10⁶ cells group exhibited the highest production efficiency (1.04 ×10 ¹² particles/million cells), approximately 7-fold and 5-fold higher than the 1.25 × 10⁶ and 5 × 10⁶ cells groups, respectively. Moreover, WJ-exNVs exhibited EV-like morphology and expression of CD63, CD81, and CD9, and promoted human dermal fibroblast proliferation in a concentration- and time-dependent manner.

Collectively, these findings provide practical insights into extrusion-processing optimization and support the potential application of WJ-exNVs as scalable EV-like nanovesicles for regenerative medicine-related research.