Dextran, albumin, and sialic acid helped preserve extracellular vesicles for a year at −20 °C
Dextran, albumin, and sialic acid helped preserve extracellular vesicles for a year at −20 °C
Researchers in China developed DDAS, a cryoprotective mixture for extracellular vesicles, which are tiny membrane particles that cells use to transport proteins and nucleic acids. After storage at −20 °C and repeated thawing, these therapeutic candidates retained more of their structure and biological activity than vesicles stored in a conventional buffer.
Extracellular vesicles are being considered as a future means of delivering stem cell signals, proteins, and RNA into the body. Such a product must travel from the laboratory to the patient, remain stable in storage, and still be the same product when it arrives. Freezing causes vesicles to aggregate, ruptures their membranes, and allows their contents to leak out.
Their effects depend on their molecular cargo: four microRNAs carried by vesicles from aging livers increased metastasis in mice. A therapeutic product must therefore retain not only the number of particles, but also the intended composition and function.
Conventional phosphate buffer does not adequately solve this problem. DMSO and glycerol can protect biological material, but they cannot remain in a product intended for direct intravenous injection. Additional purification is required after thawing.
Platelet preservation poses the same engineering problem. In a laboratory study, a low-dose DMSO protocol that required no washing recovered 94,4% of the cells after thawing. The DDAS authors tested whether vesicles could be stored in a way that allowed them to be administered after thawing without additional purification.
On July 13, the Journal of Nanobiotechnology published an early, unedited version of the DDAS study. The mixture contains dextran, albumin, and sialic acid. The authors developed it by starting with a list of molecules found in human body fluids. Generative models narrowed the list of candidates, after which the formulation was tested in vesicles, cells, and mice.
Vesicles derived from cells lining blood vessels in the brain were stored at −20 °C for a year. DDAS preserved particle numbers, RNA, and membranes better than conventional buffer. By month 12, the authors recovered about 50% of the nucleic acids. After five freeze and thaw cycles, it also outperformed both the buffer and a commercial cryoprotectant. The authors propose that dextran reduces collisions between particles, albumin forms a protective film, and sialic acid gives the vesicle surface a negative charge.
Vesicles stored in DDAS can be injected intravenously immediately after thawing. In an experiment involving six young male mice with deep burns, stem cell vesicles stored in this mixture accelerated wound closure more than the same vesicles stored in conventional buffer. By day 12, the wounds of mice treated with vesicles stored in DDAS were almost closed.
Freezing damages vesicle membranes and their cargo. According to the authors, DDAS allows the vesicles to be administered without further purification.