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Brian Paszkiet 1 , Virginia Spencer 1 , Jeff Fein 1 , Chao Yan Liu 1 , Matt Dallas 1 , Shyam Kumar 1 , Mu Li 2 , Sasha Vlassov 2 , Brian Burgin 3 , David Judd 3 , Tine Hildisch 4 , Mike Scribner 3 , and Jon Zmuda. 1 Thermo Fisher Scientific, 1 Frederick, MD, USA, 2 Austin, TX, USA, 3 Grand Island, NY, USA, and 4 Oslo, Norway RESULTS ABSTRACT Exosomes are small, 30–150 nm vesicles shed by nearly all cells of the body. Recent studies have identified numerous specialized functions of exosomes, some of which may have the potential for clinical and diagnostic use. Currently, researchers using cell culture to study exosomes still incorporate fetal bovine serum (FBS) into their culture medium, even though FBS possesses extremely high levels of bovine exosomes that can confound downstream analyses. To circumvent the problems arising from bovine exosome contamination in FBS, researchers typically deplete FBS of exosomes by ultracentrifugation. However, we have determined that this process is not very efficient, generally achieving only about 50% depletion of exosomes, while adding considerable time, effort, and variability to the cell culture workflow. An exosome-specific assay, which involves the isolation and fluorescence labeling of exosomes, was developed during this study to allow the quantification of remaining exosomes after depletion. To address the inefficiency of ultracentrifugation to deplete exosomes from FBS, we have developed a proprietary manufacturing process that achieves significantly improved levels of exosome depletion. To obtain high- quality exosome-depleted FBS, this proprietary method was used to remove bovine exosomes while still maintaining cell culture performance. Pilot and manufacturing-scale depletion studies showed ≥90% exosome depletion from FBS while maintaining cell culture performance at >90% of the source FBS (used to make the exosome-depleted FBS) for multiple cell lines commonly used in exosome research. The exosome-depleted FBS developed in this study was also validated to be depleted of exosomes by CD63 western blot analysis, RT-qPCR, nanoparticle tracking analysis, and a fluorescence staining assay. The proprietary exosome depletion process developed in this study represents a significant improvement to enable better performance when conducting exosome-related studies and downstream analyses. CONCLUSIONS We have developed an improved manufacturing process for making exosome-depleted FBS that has: • Improved exosome depletion compared to ultracentrifugation • Similar cell culture performance compared to the lot-matched source FBS • A ready-to-use format, giving improved workflows for exosome- related research REFERENCES 1. Vlassov AV et al. (2012) Biochim Biophys Acta 1820(7):940–948. 2. Labeling exosomal RNA and membrane components using fluorescent dyes. Application note available at: https://tools.thermofisher.com/ content/sfs/brochures/Exosome%20Tracing_App%20Note.pdf 3. Laulagnier K et al. (2004) Biochem J 380:161–171. 4. Li M et al. (2014) Phil Trans R Soc B 369:20130502. 5. Schageman J et al. (2013) Biomed Res Int 2013:253957.. 6. Zeringer E et al. (2013) World J Methodol 3(1):11–18. 7. Eitan E et al. (2015) J Extracell Vesicles 4:2673–2683. ACKNOWLEDGMENTS We would like to acknowledge the assistance provided by the R&D teams located at the Thermo Fisher Scientific sites in Austin, TX, Frederick, MD, and Grand Island, NY. We also thank Zhaohao Liao, Dillon Muth, and Dr. Kenneth Witwer from the Johns Hopkins University School of Medicine for sharing the data in Figure 9. TRADEMARKS AND/OR LICENSING Gibco ™ , Invitrogen ™ , Applied Biosystems ™ , NanoSight ™ , and Vi-CELL ™ products were used to produce all information presented on this poster. © 2017 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. NanoSight is a trademark of NanoSight Ltd. Vi-CELL is a trademark of Beckman Coulter, Inc. Development of an Improved Process for the Depletion of Exosomes from Fetal Bovine Serum Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • www.thermofisher.com MATERIALS AND METHODS Throughout the experiments described, Gibco ™ Fetal Bovine Serum, qualified, US origin (Cat. No. 26140079) was used. This material was subjected to exosome depletion using 2 methods: the proprietary exosome depletion method developed here, and ultracentrifugation. This material was subjected to a variety of different analyses: • The exosome depletion from FBS samples was verified by analysis on a NanoSight ™ instrument (30–150 nm) and by a fluorescence staining assay (Figure 2) • Cell culture performance was assessed, using 10% source FBS and Gibco™ Exosome-Depleted FBS (Cat. No. A25904), in several cell lines commonly used by exosome researchers (Figure 3) • FBS, before and after exosome depletion, was evaluated by SDS- PAGE (Figure 4), western blot for CD63 (Figure 5), and RT-qPCR for the common exosomal miRNA markers let7e, miR16, miR21, miR23a, miR24, and miR122 (Figure 6) • A comparison of exosome depletion efficiency was made with different manufacturing processes, including ultracentrifugation and the proprietary process developed in this study (Figure 7) Figure 1. The anatomy of an exosome. Representation of a mid-size exosome, about 60 nm in diameter, with the relative size of the membrane and cargo (blobs = proteins, green ribbons = RNAs) drawn in proportion. Additionally, the lipids in the membrane are symbolized as shown in the key (Vlassov et al. 2012). Figure 2. Exosome depletion verified by NanoSight instrument and fluorescence staining assay. Exosome depletion was verified by analysis on a NanoSight instrument (comparing the 30–150 nm count before and after exosome depletion) as well as a fluorescence-based assay. Briefly, the fluorescence assay involves extracting exosomes using the Invitrogen™ Total Exosome Isolation Reagent (FROM SERUM) (Cat. No. 4478360), and then staining the isolated exosomes with a lipophilic dye. The percent depletion is derived by comparing the fluorescence signal of the Exosome-Depleted FBS with that of the source FBS. The first two exosome-depleted lots shown above were produced by our proprietary method. Also included were an ultracentrifuged sample and a product from another supplier. The results demonstrated greater than 90% depletion of exosomes by our proprietary method, as compared to ultracentrifuged FBS and the other commercial product. Figure 3. Cell culture performance: viability and viable cell density as a percentage of source FBS. Cell lines were grown in basal medium (Gibco ™ DMEM, Cat. No. 10566016) containing 10% Exosome-Depleted FBS or source FBS, and assayed for viable cell density (VCD) and cell viability. Results are presented as the viability or VCD that was achieved in our Exosome-Depleted FBS as a percentage of that achieved in the source FBS used to make the Exosome-Depleted FBS. Figure 4. Analysis of FBS by SDS-PAGE. Equal masses (10 µg) of FBS proteins were analyzed by SDS-PAGE. Our Exosome-Depleted FBS looks very similar to the source FBS used in production, whereas the other supplier’s product shows protein band smearing. MW: molecular weight marker. 1. Source FBS, lot 1536383 2. Exosome-Depleted FBS, lot 1536383X 3. Ultracentrifuged FBS, lot 1536383 4. Source FBS, lot 1566368 5. Exosome-Depleted FBS, lot 1616564 (from lot 1566368) 6. Ultracentrifuged FBS, lot 1566368 7. Exosome-depleted FBS from another supplier 4 3 2 1 MW 7 5 6 kDa 198 98 62 49 38 28 17 Figure 5. CD63 content by western blot analysis. 1. Exosome-Depleted FBS 2. Source FBS for Exosome-Depleted FBS 3. Ultracentrifuged FBS 4. Exosome-depleted FBS from another supplier 4 3 2 1 Exosomes were precipitated from FBS samples, run by SDS-PAGE, and probed by western blotting for CD63. Compared to all other FBS samples tested, our Exosome-Depleted FBS shows no observable CD63, indicating excellent exosome depletion. Figure 6. miRNA detection by RT-qPCR in FBS- derived exosomes. Three lots of FBS were subjected to 3 different methods of exosome depletion: 110,000 × g for 3 hours (ultracentrifuge 1), 150,000 × g for 18 hours (ultracentrifuge 2), or a proprietary method (Exosome-Depleted FBS). Source FBS: prior to any exosome depletion process. All samples were subjected to a fluorescence staining assay (see Figure 2). The improvement and consistency in exosome depletion using our proprietary method, compared to ultracentrifugation, is demonstrated by the reduced fluorescence signal. Figure 8. Exosome-Depleted FBS, now available in the new 50 mL One Shot ™ format. Along with our standard 500 mL offering, Exosome-Depleted FBS is now available in our 50 mL One Shot format (Cat. No. A2720803). The new One Shot bottle has been designed to eliminate the need to aliquot FBS, minimizing contamination and saving you time. (A) Consistent with findings from Eitan et al. 2015, exosome depletion of FBS results in a modest reduction in proliferation of H9 T cells. *P < 0.002. (B) A significant increase in HIV-1 RF viral production was observed under exosome-depleted conditions, with lower levels of endogenous exosomes resulting in higher viral titers. **P < 0.03. Figure 9. Exosome-Depleted FBS supports enhanced viral production in H9 T cells. A B Figure 7. A comparison of exosome depletion methods. RNA was isolated from FBS-derived exosomes using the Invitrogen™ Total Exosome RNA & Protein Isolation Kit (Cat. No. 4478545). Levels of let7e, miR16, miR21, miR23a, miR24, and miR122 were assessed via RT-qPCR using the Applied Biosystems™ TaqMan ® MicroRNA Reverse Transcription Kit (Cat. No. 4366597). Our Exosome-Depleted FBS exhibited lower levels of all markers, as indicated by the higher C t values. Ceramide Sphingomyelin Phosphatidylserine Phosphatidylethanolamine Lysophosphatidylcholine Phosphatidylcholine Phosphatidylinositol Cholesterol 1 0.0 0.4 0.2 Absorbance Days in culture * * * 0.6 0.8 2 3 1 0 50 p24 concentration (ng/nL) Days post-infection ** ** 100 RPMI + 10% FBS RPMI + ultracentrifuge-depleted FBS RPMI + Exosome-Depleted FBS 150 2 3 Percent exosome depletion compared to source FBS Percent depletion by NanoSight measurement Exosome- depleted lot 1536383X Exosome- depleted lot 1616564 100 90 80 70 60 50 40 30 20 10 0 Ultracentrifuged Exosome- depleted FBS from another supplier Percent depletion by fluorescence assay 700 600 500 400 300 200 100 0 Relative fluorescence units Lot 1 Lot 2 Lot 3 Source FBS Ultracentrifuge 2 Exosome-Depleted FBS Ultracentrifuge 1 Percent performance compared to source FBS MCF7e HeLa Jurkat PC3 HEK 293 A549 100 90 80 70 60 50 40 30 20 10 0 VCD Viability
