Tetraspanin 4 Mediates Cholesterol-Dependent Exosome Membrane Protection from Cryodamage

Exosomes, nanosized extracellular vesicles carrying proteins, lipids, and nucleic acids, hold great potential in therapeutic applications. Cryopreservation, a widely used method for their preservation and transport, often causes irreversible damage. Understanding the molecular mechanisms underlying biomembrane resistance to cryodamage is crucial for advancing cryopreservation techniques. In this study, we find that tetraspanin 4 (TSPAN4) and other tetraspanin family proteins play an essential role in protecting exosomes from cryodamage, likely due to their role in cholesterol binding and membrane microdomain formation. Furthermore, we engineered TSPAN4-loaded exosomes, which demonstrated enhanced cryoprotection while maintaining a similar protein composition and uptake efficiency compared to wild-type exosomes. Our novel cryopreservation strategy, which does not rely on external agents, offers a promising approach for advancing the clinical translation of exosomes as therapeutic agents.