Preservation of exosomes at room temperature using lyophilization

Abstract

The application of exosomes as a therapeutic reagent or drug delivery vehicle can be expanded by developing a method to preserve exosomes. Although exosomes are generally stored at -80 °C, this temperature is not suitable for their handling or transportation and, therefore, other storage methods are desirable. Lyophilization is a promising storage method that can be used to preserve various substances at room temperature. In this study, we sought to develop a room temperature preservation method for exosomes using lyophilization and compared the properties of the lyophilized exosomes with ones stored at -80 °C. Lyophilization without cryoprotectant resulted in the aggregation of B16BL6 melanoma-derived exosomes, while the addition of trehalose, a cryoprotectant, prevented aggregation during lyophilization. PAGE analysis revealed that the proteins and RNA of exosomes were protected following lyophilization in the presence of trehalose. Lyophilization had little effect on the pharmacokinetics of Gaussia luciferase (gLuc)-labeled exosomes after an intravenous injection into mice. Moreover, it was found that lyophilized exosomes retained the activity of loaded gLuc and immunostimulatory CpG DNA for approximately 4 weeks even when stored at 25 °C. In conclusion, lyophilization with trehalose is an effective method for the storage of exosomes for various applications.

Keywords: Exosome; Freeze-drying; Lyophilization; Stability; Trehalose.