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: nonlyophilized (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), nonlyophilized EVs, or lyophilized EVs (5% sucrose). Among the preservation conditions, the 5% sucrose group retained the highest EV yield, exhibited a unimodal particle size distribution, and preserved EV 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 nonlyophilized EVs and 5% sucrose-lyophilized EVs. In the ALF model, 5% sucrose-lyophilized EVs significantly reduced serum alanine transaminase (ALT) levels, inflammatory cytokines, hepatocyte necrosis, TUNEL-positive cells, PCNA-positive cells, and Ki-67-positive cells, with effects comparable to those of nonlyophilized EVs. These findings demonstrate that lyophilization with 5% sucrose effectively preserves MSC-EV integrity and therapeutic potential, supporting future clinical applications of MSC-EVs for liver disease treatment.<b>NEW & NOTEWORTHY</b> This study establishes lyophilization with 5% sucrose as an effective preservation method for mesenchymal stem cell-derived extracellular vesicles (MSC-EVs). Preserved EVs maintained structural integrity and miRNA profiles comparable to fresh EVs. In a mouse model of acute liver failure, they demonstrated equivalent therapeutic efficacy. These findings overcome storage limitations, facilitating the clinical translation of MSC-EVs as a stable treatment for liver diseases.