Lyophilized Mesenchymal Stem Cell-Derived Extracellular Vesicles Improve Outcomes in Acute Liver Failure Models

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) are promising for the treatment of liver diseases, including acute liver failure (ALF). However, efficient preservation methods suitable for clinical use remain under investigation. In this study, we evaluated the preservation efficacy and therapeutic effects of lyophilized MSC-EVs in a mouse model of ALF. EVs were isolated from bone marrow-derived MSCs and allocated to four preservation conditions: non-lyophilized (fresh), phosphate-buffered saline (PBS), 1% sucrose, and 5% sucrose. EVs were characterized by nanoparticle tracking analysis, Bioanalyzer, absorbance measurements, RNA sequencing, and transmission electron microscopy (TEM). ALF was induced by D-galactosamine and TNF-α, and mice were treated with PBS, empty EVs (m-Encapsome), non-lyophilized EVs, or lyophilized EVs (5% sucrose). Among the preservation conditions, the 5% sucrose group retained the highest EVs yield, exhibited a unimodal particle size distribution, and preserved EVs morphology, whereas the PBS and 1% sucrose groups showed structural damage and multimodal particle size distributions. Total RNA and protein levels were comparable among groups; however, miRNA sequencing demonstrated a strong correlation between non-lyophilized EVs and 5% sucrose–lyophilized EVs. In the ALF model, 5% sucrose–lyophilized EVs significantly reduced serum ALT levels, inflammatory cytokines, hepatocyte necrosis, TUNEL-positive cells, PCNA-positive cells, and Ki-67-positive cells, with effects comparable to those of non-lyophilized EVs. These findings demonstrate that lyophilization with 5% sucrose effectively preserves MSC-EVs integrity and therapeutic potential, supporting future clinical applications of MSC-EVs for liver disease treatment.