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.

Figure 1. Purification of beta-ELVs using differential centrifugation and ultrafiltration.

Four-day supernatants from MIN6 beta-cell cultures grown in OptiMEM 1% FCS exosome-depleted media were centrifuged and filtered through a 0.2 μm filter sterilization device to remove cells and large debris. Next, supernatants were split and concentrated on an ultracel-membrane with a molecular weight cut off (MWCO) of 100 kDa and washed three times with 15 ml of either PBS or TRE. Approximately 300 μl of concentrate were recovered, re-suspended in 10 ml of wash buffer and pelleted by ultracentrifugation twice. The final ELV-pellet was recovered in approximately 1 μl of PBS or TRE per ml of culture supernatant. ELVs were stored at 4 °C for one day or at −80 °C for up to one year. ELVs were routinely assessed for particle size distribution, protein and RNA content, and biological activity.

Figure 2. TRE does not alter MIN6 insulinoma cell-derived exosome characteristics.

(A) Particle size distribution of beta-ELVs measured by nanoparticle tracking analysis. The size distribution curve fits average particle size and concentration. Data from one representative sample out of twelve are shown. (B) Western blot analysis of 15 μg of total cell lysates (MIN6) or beta-ELV proteins reveal the presence of canonical exosomal protein markers and the absence of cellular calnexin. (C,D) Experion automated electrophoresis analysis of (C) protein profiles under reducing conditions, and (D) total RNA isolated from MIN6 cells (upper panel) and of beta-ELVs in PBS or TRE (lower panel). (E) Cryo-electronic tomography images of fully hydrated, unstained beta-ELV isolated in PBS or TRE show a population of membrane vesicles of heterogeneous shape and sizes. Scale bars: 100 nm.

Figure 3. TRE reduces aggregation of beta-ELVs.

(A–E) NTA of beta-ELVs isolated using either PBS or TRE buffer; n = 12 independent samples; Wilcoxon two-tailed matched pairs test. (A) While the particle mode remained unchanged (p = 0.0566), addition of trehalose significantly decreased (B) mean particle size (***p < 0.001) and (C) standard deviation (**p < 0.01). This reduction in size paralleled an increase in (D) particle recovery and (E) the number of particles per μg of protein obtained. (F) Zeta potential was measured on a qNano instrument (Izon) on n = 6 independent samples; Wilcoxon two-tailed matched pairs test, ns (p = 0.3125).

Figure 4. TRE protects against alteration of particle structure induced by freezing.

(A,B) NTAv3.1 analysis of fresh versus freeze-thawed beta-ELVs. Main graphs depict results obtained for beta-ELVs after one or four freeze-thaw cycles (n = 7–8 biological replicates; Wilcoxon two-tailed matched pairs test). The PSD’s standard deviation and particle number recovered per ml of culture supernatant increased significantly for PBS ELVs, but remained unchanged for TRE ELVs (*p < 0.05). Graph insets show results obtained for fresh beta-ELVs or after one freeze-thaw cycle in an independent experiment (n = 5 biological replicates; Wilcoxon two-tailed matched pairs test showed no significant differences).

Figure 5. Beta-ELV uptake and immune-stimulatory activity.

(A,B) RAW264.7 macrophage suspension cultures were incubated for six hours in the presence of 20 μg/ml of freshly isolated CFDA-SE labelled beta-ELVs at 37 °C or 4 °C. Internalisation was monitored by flow cytometry analysis gating on 7-AAD negative events. (A) Clear histograms represent CFDA-SE fluorescence intensities for beta-ELVs (PBS) treated cells at 37 °C (solid line) or 4 °C (dashed line). Grey shading indicates auto-fluorescence of untreated cells. Data for one representative experiment out of five are shown. (B) Comparison of CFDA-SE positive population between beta-ELV treated cells and no treatment controls depicted as % CFDA-SE⁺ cells at 37 °C - % CFDA-SE⁺ cells at 4 °C (n = 4–5 from five independent experiments). Kruskal-Wallis test * p < 0.05, ns (p > 0.9999). (C) TNF-alpha cytokine secretion in culture supernatants of RAW264.7 macrophages stimulated with defrosted beta-ELVs for 18 h as measured by ELISA. Results are represented as mean TNF-alpha stimulation indexes (S.I.) of biological replicates ± SD (n = 7–8). Stimulation index was calculated as the difference between TNF-alpha levels measured in exosome-treated versus untreated macrophage cultures. Significant differences between PBS and TRE exosomes stimulation indexes were calculated using two-way ANOVA (****p < 0.0001 and *p = 0.0401 for doses of ELVs in μg/ml or particle count/ml, respectively). Data from one (inset) or two (main graph) representative experiments are shown.