(M0930-04-23) Beneficial Effects of Trehalose on Mesenchymal Stem Cell-Derived Exosomes

Purpose: In recent years, basic and clinical research on extracellular vesicles (EVs) including exosomes, which are natural nanoscale particles, has been intensified. However, strategies to promote the stable release of exosomes are underway. In this study, we investigated effect of trehalose on exosome release from mesenchymal stem cells (MSCs), which have been reported to have diverse medical applications, using flow cytometry (FCM) analysis and other methods with reference to MISEV2018 guidelines.

Methods: Cell culture and exosome induction
Normal human MSCs (Kurabo Industries Ltd.) were cultured in Dulbecco’s Modified Eagle Medium (Nissui Pharmaceutical Co., Ltd.) replenished with 10% supplement mix for MSC Growth Medium DXF (PromoCell™ GmbH). Cells were cultured under standard conditions in a humidified 5% CO₂ and air mixture at 37°C. After incubation for 24 hrs, the culture supernatant was removed, cells were washed with PBS (-) three times and transferred to cultures. Then, fresh culture medium containing Trehalose SG (Hayashibara Co., Ltd.) solution with or without lipopolysaccharide (LPS; Merck) was added and incubated further for 48 hrs.
Isolation of MSCs-derived exosomes
To isolate and purify the MSCs-derived exosomes, ultracentrifugation and ultrafiltration methods were examined. The concentrated, EVs in the filter cup were collected and adjusted to 200 μL using 0.1 μm filtered PBS as the exosome fraction. Protein levels were determined using a Pierce™ BCA protein assay kit (Thermo Fisher Scientific Inc.).
Stimulation of THP-1 macrophages
After the resting period, THP-1 macrophages were washed once with RPMI-1640 medium supplemented with 1% FCS and 1% Penicillin-Streptomycin and were incubated with various concentrations of exosome solution. After the incubation period, THP-1 macrophages were stimulated with 20 ng/mL human IFN-γ and 5 µg/mL LPS at 37°C for 24 hrs.
Analysis of exosomes by surface antigen staining and FCM
Referring to the article by Robert S. et al., EVs or exosomes purified from MSCs (Passage 4-10) culture supernatants by ultracentrifugation or ultrafiltration were characterized by FCM (Gallios AS08042 Beckman Coulter Inc.) analysis using antibodies specific for CD9, CD63 and CD81 antigens.
Evaluation of TNF-α production by THP-1 cells as exosome activity
Levels of human TNF-α in culture supernatants were measured by two-site sandwich ELISA as described.

Results: ・FCM-based assay was performed for characterization and the counting of MSC-derived exosomes. Under the conditions in our experiment, the fluorescent beads for FCM standardization (0.1 μm) were visible on the plot and the Flow-Count™ standard particles for counting (10 μm diameter) were plotted at a position (gate T) where they did not overlap with the exosomes (Figure 1A and B). Exosome surface marker analysis revealed that EVs released from MSCs were 99.6%, 99.8%, and 99.9% positive for the exosome markers CD9, CD63, and CD81 antigens, respectively (Figure 1C).
・Next, the effect of trehalose on exosome production by MSCs was investigated. Various concentrations of trehalose were added to MSCs culture medium, and the cell number was counted after 48 hrs incubation as shown in Figure 2A. Addition of trehalose (10, 50 and 100 mM) had no effect on the MSCs numbers (Figure 2A), nor did they cause any obvious cytotoxicity. FCM analysis of the number of EVs in cell culture supernatants showed that the addition of trehalose resulted in dose-dependent increase in the number of EVs, with statistically significant differences at 50 mM and 100 mM compared with control culture (Figure 2B, n=3). The number of exosomes after purification by ultracentrifugation was also shown to increase with the addition of 50 mM and 100 mM trehalose (Figure 2C).
・Exosomes produced by MSCs have been reported to exhibit anti-inflammatory effects by inducing differentiation of macrophages from M1- to M2-type when cultured under inflammatory conditions using LPS and are expected to be utilized for wound healing and immunotherapy. Therefore, we evaluated the anti-inflammatory effects of MSC-derived exosomes using TNF-α production from polarized THP-1 macrophages as an indicator. Interestingly, MSC-derived exosomes induced by LPS showed a dose-dependent inhibition of TNF-α production, while exosomes derived from MSCs alone showed a statistically significant enhancement of TNF-α production (Figure 3A). Furthermore, trehalose did not affect the anti-inflammatory effects of MSCs-derived exosomes induced by LPS, suggesting that trehalose does not affect the desired physiological function of exosomes (Figure 3B).

Conclusion: Bosch S. et al. reported that trehalose induced MIN6 insulinoma cells to release higher particle counts compared with those of PBS using nanoparticle tracking analysis. Together with these results, we demonstrated here for the first time that addition of trehalose not only increases the number of EVs in MSCs culture supernatant, but also significantly enhances exosome production. We further demonstrated that the addition of trehalose during exosome production by MSCs increases the production of exosomes without compromising their function and contributes to the prevention of reduction in the residual exosomes during purification. In addition, our results provide a very promising strategy toward industrial resolution of exosomes.
Figure 1. Characterization of MSCs-derived exosomes by flow cytometric analysis.

Figure 2. Effects of trehalose on MSC count (A), EV production (B) and exosome yields (C).

Figure 3. Dose-dependent anti-inflammatory effects by MSCs-derived exosomes induced by LPS (A) and effect of trehalose on its action (B).