Trehalose prevents aggregation of exosomes and cryodamage

Abstract

Exosomes are important mediators in intercellular communication. Released by many cell types, they transport proteins, lipids, and nucleic acids to distant recipient cells and contribute to important physiopathological processes. Standard current exosome isolation methods based on differential centrifugation protocols tend to induce aggregation of particles in highly concentrated suspensions and freezing of exosomes can induce damage and inconsistent biological activity. Trehalose is a natural, non-toxic sugar widely used as a protein stabilizer and cryoprotectant by the food and drug industry. Here we report that addition of 25 mM trehalose to pancreatic beta-cell exosome-like vesicle isolation and storage buffer narrows the particle size distribution and increases the number of individual particles per microgram of protein. Repeated freeze-thaw cycles induce an increase in particle concentration and in the width of the size distribution for exosome-like vesicles stored in PBS, but not in PBS 25 mM trehalose. No signs of lysis or incomplete vesicles were observed by cryo-electron tomography in PBS and trehalose samples. In macrophage immune assays, beta-cell extracellular vesicles in trehalose show consistently higher TNF-alpha cytokine secretion stimulation indexes suggesting improved preservation of biological activity. The addition of trehalose might be an attractive means to standardize experiments in the field of exosome research and downstream applications.

Developmental competence of in vitro matured ovine oocytes vitrified in solutions with different concentrations of trehalose

“…Our experiment also revealed that vitrification in the absence of trehalose (0.0 M) impairs developmental competence in vitri- (Bosch et al, 2016) and stabilizing the structural and functional integrity of cell membrane in lowwater media (Kaushik & Bhat, 2003;Olsson, Jansson, & Swenson, 2016…”

“…Our findings suggest that the detrimental effects of CPAs on ZP were remarkably reduced in the presence of trehalose. Trehalose has been recently found to be useful in preventing the cryodamage (Bosch et al, ) and stabilizing the structural and functional integrity of cell membrane in low‐water media (Kaushik & Bhat, ; Olsson, Jansson, & Swenson, ) . The results of several studies show that stabilizing effects of sugars in general and trehalose in particular, are related to a specific interaction of sugars with biological molecules in bilayer membranes (Jain & Roy, ; Konov et al, ; Olsson et al, ).…”

Hypoxia promotes IL-32 expression in myeloma cells, and high expression is associated with poor survival and bone loss

“…We also observed that a fraction (;50%) of IL-32 was secreted as a free, nonvesicular protein. Whether this is due to disruption of vesicles and release of IL-32 47 or whether IL-32 is also released from the cells as a free protein remains to be investigated.…”

Exosomes purified from a single cell type have diverse morphology and composition

“…Exosomes are commonly considered to be a homogeneously shaped group of vesicles, sized 40-100 nm in diameter (20). On the other hand, an abundance of morphological diversity has emerged when extracellular vesicles from bodily fluids (11-13, 15, 16) and cell culture supernatant (29)(30)(31) were studied using cryo-electron microscopy (cryo-EM), a method which allows visualization of membrane bilayers and internal features (11)(12)(13)31). However, thorough morphological studies using cryo-EM on exosomes purified from a single cell type are lacking to date.…”