Effect of pH, temperature and freezing-thawing on quantity changes and cellular uptake of exosomes

Abstract

Dear Editor, Exosomes are cup-shaped small (30–150 nm) extracellular vesicles with the structure of lipid bilayer membrane (Tkach and Thery, 2016) containing proteins, mRNAs and microRNAs that mediate intercellular communication (Valadi et al., 2007). Unlike other extracellular vesicles, exosomes are released into the extracellular space when the multivesicular bodies (MVBs) fuse with the plasma membrane (Colombo et al., 2014). Almost all cell types can secret exosomes and exosomes exist in diverse biological fluids, such as blood, urine, saliva, hydrothorax and breast milk (Thery et al., 2006). Up to now, a number of studies have demonstrated the functions of exosomes in disease development and the potential clinical applications in diagnosis and therapy (Shao et al., 2016). To conduct reproducible studies on exosomal content and function, storage conditions need to have minimal impact on exosomes. There have been a few studies providing partial confirmation of the effect of different storage conditions on exosomes currently. Using exosomes from urine (Zhou et al., 2006) and conditioned medium (Lee et al., 2016) respectively to investigate the influence of storage temperature on exosomes as measured by Western blot, both groups have concluded that storage below −70 °C for a long time is the best temperature for the recovery of exosomes. On the other hand, Sokolova et al. (2011) applied nanoparticle tracking analysis (NTA) to measure the size changes of exosomes at different temperatures, revealing that storage at 37 °C led to more reduction in exosome sizes than that at 4 °C. However, in this study no information about changes in the particle concentration was reported. Some other studies revealed the effect of pH, storage temperature and cycles of freezing and thawing only on the yield of exosome isolation, but not on quantity changes during storage (Akers et al., 2016; Ban et al., 2015; Zhao et al., 2017). Therefore, the standard criterion of exosomal preservation condition is still undefined. Herein, we used HEK 293T cells and ExtraPEG method (Rider et al., 2016) to investigate the influence of multiple storage conditions (temperature, cycles of freezing and thawing, pH) on the quantity changes and cellular uptake of exosomes. ExtraPEG is a new polyethylene glycol (PEG) precipitation method for the purification exosomes without affecting their biological activity. Generally, ultracentrifugation (UC) (Mincheva-Nilsson et al., 2016) is most reliable but time-consuming; and precipitation methods such as ExoQuick (patent number: US20130337440 A1) and ExtraPEG can obtain higher yields of exosomes but with impurity of co-precipitated proteins. First, exosomes from the conditioned medium were extracted by ExtraPEG or UC method. After isolation, transmission electron microscope (TEM), NTA and Western blot were performed to analyze exosomes. Exosomes extracted by UC or ExtraPEG were similar in cup-shaped structure (Fig. S1A and S1B), size distribution (Fig. S1C and S1D). And as representative exosome biomarkers, ALG-2-interacting protein X (ALIX), heat shock protein 70 (HSP70) and tumor susceptibility gene 101 (TSG101) were detected in exosomal protein while β-tubulin, widely used as an internal reference to analyze intracellular protein levels, was not detected in exosome samples (Fig. S1E and S1F). These data indicated exosomes were successfully isolated by ExtraPEG method and suitable for the following experiments. After isolation, the exosome pellets were divided equally into several portions and each portion was stored at different temperatures (−80 °C, −20 °C, 4 °C, 37 °C and 60 °C), or through 1–5 cycles of freezing to −80 °C and thawing, or at different pH levels (pH 4, pH 7 and pH 10). After 24 h, NTA and Western blot were performed to measure the remaining quantity of exosomes. Regarding temperatures, the exosomes stored at 4 °C had the highest concentration (Fig. 1A). Consistent with the NTA results, the exosomes stored at 4 °C showed higher levels of representative exosome markers ALIX, HSP70 and TSG101 (Fig. 1B). With the increasing cycles of freezing and thawing, the exosomal concentration and protein levels of ALIX, HSP70 and TSG101 all decreased (Fig. 1D and 1E). For different pH levels, the loss of exosomal concentration and three exosome markers ALIX, HSP70 and TSG101 at pH 4 and pH 10 was more than that at pH 7 (Fig. 1E and 1F). Interestingly, exosomes stored at pH 4 decreased more sharply than that at pH 10 (Fig. 1F and 1G), suggesting that acidic environment is more destructive for the exosome stability. The size data (Tables S1–3) showed that there was no significant difference in exosome size between different temperature groups, as well as different cycles of freezing and thawing and different pH groups, which indicated that exosomes are not discomposed into smaller or fused into larger vesicles but largely degraded during storage. Next the levels of exosome-associated proteins for the lo