An assessment of the performance and effectiveness of collagenase types in quality improvement of small extracellular vesicles from human adipose-derived stromal vascular fraction for precision medicine applications

Introduction: Small extracellular vesicles (sEVs) are gaining recognition as promising tools for medical applications due to their natural compatibility with biological systems and potential for targeted delivery. Among the various sources, tissue-derived sEVs (ti-sEVs) offer unique properties compared to vesicles from other origins. However, the dense extracellular matrix (ECM) within tissues may trap sEVs, limiting their yield and purity. These limitations remain major obstacles to clinical translation, as therapeutic efficacy depends on abundant, high-quality vesicles capable of efficient cargo transfer. Efficient isolation strategies are therefore critical. Methods: In this study, we focused on the stromal vascular fraction (SVF) of healthy human adipose tissue (hHAT), a heterogeneous cell mixture that retains properties of its tissue of origin. SVF is typically isolated by enzymatic digestion; yet, the impact of different collagenases on sEV quality and yield is unclear. To address this, we examined the effects of collagenase type II and type IV on SVF cells and their release of sEVs in vitro. SVF-sEVs were quantified after 16–24 h of conditioning and characterized by standard assays—including cell-viability assays, nanoparticle tracking analysis, transmission electron microscopy, fluorescence microscopy, and western blotting—following MISEV guidelines. Result: Across six hHAT-SVF samples, collagenase type II produced a higher yield (6.94 particles/ml vs. 4.55 particles/ml with type IV) while preserving vesicle integrity and reducing contamination. Conclusion: These findings indicate that collagenase type II is a gentler and more effective option, significantly enhancing SVF-sEV yield and quality.