Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Sep 30:3.
doi: 10.3402/jev.v3.24783. eCollection 2014.

Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum

Ganesh Vilas Shelke  1 , Cecilia Lässer  1 , Yong Song Gho  2 , Jan Lötvall  1
Affiliations

Importance of exosome depletion protocols to eliminate functional and RNA-containing extracellular vesicles from fetal bovine serum

Ganesh Vilas Shelke et al. J Extracell Vesicles. .

Abstract

Extracellular vesicles (EVs), including the nano-sized exosomes, have the capacity to transfer multiple functional molecules between cells. In cell culture experiments, fetal bovine serum (FBS) is often used to supplement cell culture medium as a nutrient, but it is important to know that the FBS also contain significant quantities of EVs. The aim of the current study was to determine whether the FBS EVs can influence cultured cell phenotype, and secondly to determine the efficiency of FBS-EV elimination protocols. Firstly, FBS that had not been depleted of EVs induced a migratory phenotype in a lung cancer epithelial cell line (A549 cells), an effect that could be mimicked by isolated FBS EVs alone. FBS-derived EVs also contained RNA, which was protected from consecutive proteinase K and RNase A treatment. Comparison of common isolation protocols suggested that an 18-hour centrifugation period eliminates approximately 95% of RNA-containing FBS EVs, whereas a 1.5-hour protocol is insufficient. In conclusion, this study shows that FBS EVs substantially influence cultured cell behaviour, but also that they can be virtually removed by an 18-hour ultracentrifugation protocol.

Keywords: Depletion protocol; Exosomes; Extracellular vesicles; Fetal bovine serum; RNA.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Depletion of extracellular vesicles reduces FBS-induced cell migration. Medium was either supplemented with 10% FBS (Medium, non-depleted FBS) or 10% FBS that had been centrifuged for 18 hours to eliminate extracellular vesicle (Medium, EV-depleted FBS). As a control, FBS-free media was used (Plain medium). A549 cells were seeded on one side of a gelatin coated membrane of a Boyden chamber to evaluate cellular transmigration towards the various stimuli on the other side of the membrane. Student's t-test was used to determine significant differences, p<0.05, ***p<0.001. n=3.
Fig. 2
Fig. 2
Characterization of FBS-derived extracellular vesicles. FBS-derived vesicles were collected at 120,000×g for 18 hours, dissolved in PBS and re-pelleted at 120,000×g for 70 minutes. a) To visualize if FBS had vesicular structures present, TEM was performed on the EV-enriched pellet. The scale bar represents 200 nm. b) Immuno-blotting for the exosome enriched proteins; TSG101, CD81, and CD63. Pellet of EVs were obtained from different batch of FBS. Cell lysate from human mast cells (HMC-1) and mouse fibroblast (NIH-3T3), as well as HMC-1 derived exosomes were used as positive controls. One hundred microgram of protein was loaded per well for all samples. c) RNA was isolated using the miRCURY total RNA isolation kit and quantified with a Bioanalyzer. d) The FBS-derived EV-enriched pellet was incubated with RNase A (0.5 µg/µl) alone (i) or was incubated with proteinase K (0.05 µg/µl) prior to the RNase treatment (ii) to study mode of RNA protection. Cellular RNA was used as control for the RNase activity (iii). Graphs are representative of the RNA profile obtained with a Bioanalyzer from two independent experiments. e) PKH67-labelled FBS-derived EVs were incubated with 7×104cells (A549) for 6 or 20 hours and visualized with a fluorescent microscope (40X). PBS was used as control for determining the PKH67 background labelling.
Fig. 3
Fig. 3
FBS-derived extracellular vesicles induce migratory phenotype in an epithelial cell line. FBS-derived EVs were collected at 120,000×g for 18 hours, washed in PBS and re-pelleted for 70 minutes at 120,000×g. Isolated vesicles were incubated with A549 cells, but were separated by a gelatin coated membrane in a Boyden chamber to evaluate cellular transmigration towards variable EVs concentrations. FBS-derived EVs showed a dose-dependent response in inducing migration in A549 cells. Kruskal-Wallis test was used to determine significant differences. Comparisons were only made against the control. *p<0.05, **p<0.01, ***p<0.001. n=3. Spearman Rank gave a significant correlation of Rs=0.82.
Fig. 4
Fig. 4
Analysis of FBS vesicles depletion protocols. EV-depleted FBS was obtained from 1.5 and 18 hours of ultracentrifugation and was used to prepare complete media (10% FBS). The media was never in contact with cells, but used to isolate exosomes from. The RNA was extracted from the 120,000×g pellet and the concentration and nucleotide size was determined by a Bioanalyzer and the Pico chip. a) Data represent the overlay of RNA profile concentration of bovine RNA retained in complete media supernatant and pelleted during exosome isolation. b) Efficiency of exosome depletion with different centrifugation period is compared based on total RNA content in their exosome preparation in complete media. Results represent mean±SEM of sample obtained from 3–6 independent experiments. c) Presence of vesicular structures was determined by transmission electron microscopy. Vesicles are from the pellet preparation from EV-depleted FBS after 18 hours of ultracentrifugation (Bar represents 200 nm).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, Melief CJ, et al. B lymphocytes secrete antigen-presenting vesicles. J Exp Med. 1996;183:1161–72. - PMC - PubMed
    1. Denzer K, van Eijk M, Kleijmeer MJ, Jakobson E, de Groot C, Geuze HJ. Follicular dendritic cells carry MHC class II-expressing microvesicles at their surface. J Immunol. 2000;165:1259–65. - PubMed
    1. Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol. 2013;200:373–83. - PMC - PubMed
    1. Subra C, Grand D, Laulagnier K, Stella A, Lambeau G, Paillasse M, et al. Exosomes account for vesicle-mediated transcellular transport of activatable phospholipases and prostaglandins. J Lipid Res. 2010;51:2105–20. - PMC - PubMed
    1. Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007;9:654–9. - PubMed