INTRODUCTION 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, 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. To address these issues, we have developed an exosome-depleted FBS using a proprietary manufacturing process, which achieves three main goals: (1) provides a more consistent cell culture reagent (compared to ultracentrifuged FBS) for exosome research, (2) improves exosome workflows, removing the need for researchers to prepare their own exosome-depleted FBS, and (3) complements our existing exosome analysis reagents. To obtain high-quality exosome-depleted FBS, a 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 lot (used to make the exosome-depleted product) for multiple cell lines commonly used in exosome research. The manufacturing process developed for this product has allowed for the creation of a product that outperforms existing offerings on the market and ultracentrifuged FBS by providing researchers with an off-the-shelf FBS that is highly depleted of bovine exosomes. Gibco™ Exosome-Depleted FBS gives the exosome researcher a more consistent FBS with improved workflows.

Brian J. Paszkiet, Virginia Spencer, Jeff Fein, Chao Yan Liu, Matt Dallas, Shyam Kumar, Mu Li, Sasha Vlassov, Brian Burgin, David Judd, Tine Hildisch, Mike Scribner, and Jonathan Zmuda, Thermo Fisher Scientific, 7335 Executive Way, Frederick, MD, USA 21704

RESULTS Exosome Depletion Verified by NanoSight Measurement and Fluorescent Staining Assay

Figure 3. 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 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 fluorescent signal of the exosome-depleted FBS with 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 competitor’s product. The results demonstrated ≥90% depletion of exosomes by our proprietary method, as compared to ultracentrifuged FBS and the competitor’s product.

Cell Culture Performance: Viability and Viable Cell Density as a Percentage of Source FBS

Figure 4. Cell lines were grown in basal medium (Gibco™ DMEM, High Glucose, Gibco™ GlutaMAX™ Supplement (Cat. No. 10566-016)) containing 10% Gibco Exosome-Depleted FBS (Cat. No. A25904) or source Gibco™ FBS (Cat. No. 26140-079), and assayed for viable cell density (VCD) and cell viability by Vi-CELL™ analysis. Results are presented as the viability or VCD that was achieved in Gibco Exosome-Depleted FBS as a percentage of that achieved in the source FBS used to make the exosome-depleted FBS.

CD63 Content by Western Blot Analysis

Figure 6. Exosomes were precipitated from FBS samples, run by SDS-PAGE, and probed by western blotting for CD63. MW: molecular weight marker. Compared to all other FBS samples tested, our Exosome-Depleted FBS shows no observable CD63, indicating excellent exosome depletion.

REFERENCES 1. Vlassov AV, Magdaleno S, Setterquist R, Conrad R (2012) Exosomes: Current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials. Biochim Biophys Acta 1820(7):940–948. 2. Labeling exosomal RNA and membrane components using fluorescent dyes. Application note available at: https://tools.lifetechnologies.com/content/sfs/brochures/Exosome%20Tracing_App%20Note.pdf 3. Laulagnier K, Motta C, Hamdi S, Roy S, Fauvelle F, Pageaux JF, Kobayashi T, Salles JP, Perret B, Bonnerot C, Record M (2004) Mast cell- and dendritic cell-derived exosomes display a specific lipid composition and an unusual membrane organization, Biochem J 380:161–171. 4. Li M, Zeringer E, Barta T, Schageman J, Cheng A, Vlassov AV (2014) Analysis of the RNA content of the exosomes derived from blood serum and urine and its potential as biomarkers. Phil Trans R Soc B 369: 20130502, published 18 August 2014. 5. Schageman J, Zeringer E, Li M, Barta T, Lea K, Gu J, Magdaleno S, Setterquist R, Vlassov AV (2013) The Complete Exosome Workflow Solution: From Isolation to Characterization of RNA Cargo. BioMed Research International Vol. 2013, Article ID 253957, 15 pages. 6. Zeringer E, Li M, Barta T, Schageman J, Pedersen KW, Neurauter A, Magdaleno S, Setterquist R, Vlassov AV (2013) Methods for the extraction and RNA profiling of exosomes. World J Methodol 3(1):11–18.

ACKNOWLEDGMENTS We would like to acknowledge the assistance provided on this project by the R&D teams located at the Thermo Fisher Scientific Austin, TX, Frederick, MD and Grand Island, NY sites.

TRADEMARKS/LICENSING © 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license. Vi-CELL is a trademark of Beckman Coulter.

FURTHER INFORMATION

thermofisher.com/exosomes

http://exosomestalk.com/index.php

Gibco Exosome-Depleted FBS: The Consistent Choice for Your Exosome Research

Thermo Fisher Scientific • 5791 Van Allen Way • Carlsbad, CA 92008 • lifetechnologies.com

Analysis of FBS by SDS-PAGE

Figure 5. Equal masses (10 µg) of FBS proteins were analyzed by SDS-PAGE. MW: Molecular weight marker. Our Exosome-Depleted FBS looks very similar to the source FBS used in production, whereas the competitor’s product shows protein band smearing.

miRNA Detection by qRT-PCR in FBS- Derived Exosomes

Figure 7. Exosomes were isolated from FBS samples, and total RNA was isolated using the Total Exosome RNA & Protein Isolation Kit (Cat. No. 4478545). RNA was analyzed by qRT-PCR for Let7e, miR16, miR21, miR23a, miR24, and miR122 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 Ct values.

Additional Products: Product Name (Cat. No.) Exosome-Human CD9 Isolation Reagent (from cell culture) (10614D)

Total Exosome Isolation Reagent (from serum) (4478360)

Exosome-Human CD81 Isolation Reagent (from cell culture) (10616D)

Exosome Immunoprecipitation Reagent (Protein G) (10612D)

Exosome-Human EpCAM Isolation Reagent (from cell culture) (10618D)

Exosome Immunoprecipitation Reagent (Protein A) (10610D)

Exosome-Human CD9 Flow Detection Reagent (from cell culture) (10620D)

Exosome-Streptavidin Isolation/Detection Reagent (10608D)

Exosome-Human CD81 Flow Detection Reagent (from cell culture) (10622D)

Exosome-Human CD63 Isolation/Detection Reagent (from cell culture media) (10606D)

Exosome-Human EpCAM Flow Detection Reagent (from cell culture) (10624D)

Exosome Spin Columns (MW 3000) (4484449)

Exosome Anti-CD9 Antibody (for western blot) (10626D)

Total Exosome Isolation Kit (from plasma) (4484450)

Exosome Anti-CD63 Antibody (for western blot) (10628D)

Total Exosome Isolation Reagent (from urine) (4484452)

Exosome Anti-CD81 Antibody (for western blot) (10630D)

Total Exosome Isolation Reagent (from other body fluids) (4484453)

Total Exosome RNA & Protein Isolation Kit (4478545)

SYTO™ RNASelect™ Green Fluorescent cell Stain - 5 mM Solution in DMSO (S32703)

Total Exosome Isolation Reagent (from cell culture media) (4478359) BODIPY™ TR Ceramide (D7540)

CONCLUSIONS We have developed an exosome-depleted FBS for your exosome research that is: •  Ready to use

•  Verified for exosome depletion

•  Verified for cell culture performance, with lot-matched source FBS available

1.  Source FBS, Lot 1536383

2.  Gibco™ Exosome- Depleted FBS, Lot 1536383X

3.  Ultracentrifuged FBS, Lot 1536383

4.  Source FBS, Lot 1566368

5.  Gibco™ Exosome- Depleted FBS, Lot 1616564 (from 1566368)

6.  Ultracentrifuged FBS, Lot 1566368

7.  Competitor’s exosome-depleted FBS

MATERIALS AND METHODS Throughout the experiments described, Gibco Fetal Bovine Serum, qualified, US origin (Cat. No. 26140-079) was used. This material was subjected to exosome depletion using 2 methods: our proprietary exosome depletion method as well as ultracentrifugation at 110,000 x g for 2 hours at 4°C. 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 fluorescent staining assay (Figure 3)

•  Cell culture performance was assessed, using 10% source FBS and Exosome-Depleted FBS, in several cell lines commonly used by exosome researchers (Figure 4)

•  FBS before and after exosome depletion was evaluated by SDS-PAGE (Figure 5), western blot for CD63 (Figure 6), and qRT-PCR for the common exosomal miRNA markers Let7e, miR16, miR21, miR23a, miR24, and miR122 (Figure 7)

The Anatomy of an Exosome

Figure 1. 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).

Gibco Exosome-Depleted Fetal Bovine Serum

Figure 2. Representative packaging and format for Gibco Exosome-Depleted FBS. Lot-matched Source FBS used to make the exosome-depleted product will also be available.

Exosome- Depleted

FBS

Lot-matched source FBS

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For Research Use Only. Not for use in diagnostic procedures.

4 3 2 1 MW 1.  Gibco™ Exosome-

Depleted FBS 2.  Source FBS for

Gibco™ Exosome-Depleted FBS

3.  Ultracentrifuged FBS 4.  Competitor’s

exosome-depleted FBS

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