What Is Lyophilization?

Lyophilization or freeze drying is the process of removing water from a frozen sample under vacuum. Water is removed via sublimation, resulting in a dry, water-free product that can be stored for extended periods of time. Lyophilization is used for the storage of proteins, tissues and even foods.

During the process of sublimation proteins can lose their structure and become damaged. Exosomes that are lyophilized with no cryoprotectant rupture, leak their contents, lose their 3D spherical shape and lose their function. To stop this damage the use of cryoprotectants such as trehalose can preserve the structure and viability of exosomes during the lyophilization process. Trehalose is a sugar molecule made up of two glucose molecules. During the sublimation as water is removed from the sample the trehalose molecule acts as a stabilizer to help retain the 3D structure of the exosomes. There are many research studies that demonstrate the beneficial effects of lyophilizing with trehalose where exosomes have been stored for long periods of time and still retain their structure and function once reconstituted.

Benefits of Lyophilization

Removing water from a solution of exosomes significantly increases the shelf life and stability of the exosomes over time. Alternative methods for exosome storage are freezing at -80°C; this has demonstrated promising results but requires specialist equipment and shipping using dry ice making this not feasible for the cosmetic market. Freezing exosomes in solution at -20 addresses the specialist equipment but has demonstrated extremely low recovery of exosomes after thawing (REF). This therefore is considered a far from idea method for exosome storage within the extracellular vesicle community. Storing exosomes in a refrigerator at 4°C is extremely practical but data demonstrates a significant and rapid loss of intact vesicles over time. Therefore, the 10B exosomes that are added to each vial would rapidly decrease therefore significantly reducing the effect the lesser number of exosomes would have when applied to the skin.

Lyophilization overcomes these limitations by allowing long term storage (9 month shelf life) of exosomes with little reduction in exosome number once the treatment is applied. Storing the lyophilized exosomes in a refrigerator at 4°C adds an additional level of control in minimizing any temperature fluctuations that may cause exosome degradation.