Introduction: Mesenchymal stromal cells (MSCs) have emerged as a novel therapeutic strategy for liver cirrhosis, demonstrating anti-fibrotic effects. We have previously shown that MSC-derived extracellular vesicles (EVs) contribute to these effects, primarily through modulation of macrophage function. In this study, we present the mechanisms underlying these effects and our progress toward clinical application.

Methods: We evaluated the therapeutic impact of MSC-derived EVs using a carbon tetrachloride induced mouse model of liver fibrosis. In vivo imaging was used to track EVs distribution, and in vitro assays assessed macrophage polarization. To enable large-scale EVs production for clinical use, we employed tangential flow filtration (TFF) and compared its yield and therapeutic efficacy to conventional ultracentrifugation. Furthermore, we investigated changes in MSCs quality and EVs induced by the number of MSCs passages by analyzing gene expression changes in MSCs using single-cell analysis and conducting omics analysis of EVs-containing factors. Changes in the effects of MSCs and EVs were verified by validating macrophage polarization changes in vitro.

Results: MSC-derived EVs promoted macrophage polarization toward a tissue-reparative phenotype in vitro and preferentially accumulated in hepatic macrophages in vivo. TFF increased EVs yield by three-fold and reduced processing time by 75% compared to ultracentrifugation, while maintaining equivalent therapeutic efficacy in the liver fibrosis model. Passage-dependent changes were observed in MSCs gene expression and EVs cargo composition, including protein and miRNA profiles, correlating with diminished macrophage-modulating capacity. Single-cell analysis revealed gene expression patterns associated with cellular senescence and degradation in late-passage MSCs.

Conclusion: MSC-derived EVs ameliorate liver fibrosis by modulating macrophage phenotype. This study highlights key advancements in the scalable production and quality assessment of EVs, laying the groundwork for their clinical application in liver fibrosis therapy.