News

Aug 23, 2016

Join the SELECTBIO Extracellular Vesicles Virtual Event made available from September 21st, and hear leading researchers talk about topics within EV biology and therapeutic potential!

 

Information

General information, events and/or links.

 

 

Regional Research Network on Extracellular Vesicles

Jan 19, 2016

Four Considerations for Enriching Extracellular Vesicles!


The lack of standardized methods is a significant bottleneck to accurately elucidating the function of EVs.

Written by Amedeo Cappione III, Ph.D, Chandreyee Das, Ph.D.

 

Four Considerations for Enriching Extracellular Vesicles

Source: http://www.genengnews.com/insight-and-intelligence/four-considerations-for-enriching-extracellular-vesicles/77900596/, published online January 18th 2016. © 2016 Genetic Engineering &
Biotechnology News:

 

Given the importance of exosomes and other extracellular vesicles (EVs) in therapeutic development and mechanistic studies of cancer, neuroscience, and immunology, there are many researchers seeking the “best” protocol for isolating these vesicles. On ResearchGate.net alone, over a hundred questions related to exosome isolation have been posted, with some threads viewed over 10,000 times.

The past year has witnessed rapid changes in EV research, as studies uncover differences in function and cargo between extracellular particle subtypes. In particular, the International Society for Extracellular Vesicles (ISEV) has published a number of recommendations for EV research methods because the lack of standardized methods for EV enrichment and characterization represents a bottleneck to accurately elucidating the function of EVs.1,2

Our survey of EV-related research presented at the Annual Meetings of the American Association for Cancer Research (AACR) demonstrated that while, in 2014 a large number of researchers were using ultracentrifugation, by 2015 the majority are using sucrose gradient fractionation. We saw few, if any, instances in which precipitation methods were used for EV enrichment. Finally, we observed an increase in the thoroughness with which EV preps were being characterized, including Western blots for positive and negative EV markers.

Research Trends Driving Need for EV Enrichment: EVs Shape Cell Environment and Carry exRNAs

In presentations at recent conferences, including the AACR meetings and the 2014 meeting of the American Society for Exosomes and Microvesicles, the most commonly cited reason for studying EVs is that they mediate cell-cell communication. EVs carry out this function via diverse mechanisms, such as cell-independent processing of miRNAs by exosomes, stroma remodeling by vesicle-borne proteases, and suppression of immune activity in the tumor microenvironment.3,4,5

 

The role of EVs in transporting extracellular RNAs (exRNAs) is also of great interest, although recent reports suggest the possibility that circulating miRNAs may not necessarily be contained within EVs.6 The National Cancer Institute has funded an exRNA research program, out of which has emerged a useful web resource compiling protocols for EV enrichment and RNA isolation. Concurrently, the NIH Extracellular RNA Communication Consortium agrees that in order to accurately assess exRNA function, there is a need to work with more pure, well-characterized, and relatively quantified preparations of EVs.7

 

Four Considerations for Designing an EV Enrichment Protocol

  •  
    Figure 1. Rapid centrifugal ultrafiltration protocol for enriching EVs from low protein samples (sera-free media, urine). Supernatant was harvested from MDA cells following 48 hour serum starvation. An aliquot was processed via ultrafiltration; the resulting fraction was labeled with Bodipy® FL Dye (Thermo Scientific). Labeled microvesicles (20% load – 10 mL starting supernatant) were spiked into a second purification (40 mL) performed by either ultrafiltration (UF), ultracentrifugation (UC), or precipitation [PPT -ExoQuick-TC™ Solution (System Biosciences)]. Recovered fractions were assayed for exosome recovery (CD63 Bead capture/CD9 flow cytometry), and Western Blotting (UF and UC only). Over 90% of the spiked EVs were recovered using ultrafiltration and ultracentrifugation. The precipitation method generated much lower yields (1A). Western blotting showed that EV-specific markers were detected in the ultrafilter retentate and in the ultracentrifugation pellet (1B).

     

     

    1. What is the source of your sample?
    Cell culture supernatant
    . If the EV source is cell culture supernatant, there are many options for preparation. One of the most popular methods is density gradient ultracentrifugation. A sample protocol would involve overlaying precleared, concentrated cell culture supernatant onto a solution of sucrose- or iodixanol-based density gradient medium. Ultracentrifugation at 100,000 x g for 2-3 hours, followed by a wash step, has been shown to provide >1 g/mL EVs, with relatively high purity (as measured by particle-to-protein ratio and absence of non-EV proteins (such as Ago2).8,9

    Affinity purification using beads conjugated to antibodies specific to vesicle-specific proteins (such as anti-CD63, anti-CD9, or anti-MHC beads) are also quite effective for cell culture supernatant samples.10 However, current affinity-based approaches lack efficient ways to elute EVs from the solid phase, and are mainly used for surface phenotyping and cargo analysis.

    Size exclusion-based purification, either by ultrafiltration, tangential flow filtration, or chromatography, may be a simple, versatile, cost-effective option for vesicle enrichment from cell culture supernatant (Figure 1). Although exact yields and purity may be dependent on the cell type and cell culture conditions, some studies show that modern ultrafiltration systems are faster and may be higher-yielding than density gradient methods.11 Another advantage is that, given the ultrafiltration device’s capacity for buffer exchange and sample concentration, purified fractions can be easily formatted to meet the requirements of any downstream analysis platform.

    Proteinaceous biological samples (Serum, Plasma, CSF). EVs are found in nearly every biological fluid, as listed in a recent review of EV function, ranging from blood and breast milk to bile and synovial fluid.12 Bodily fluids are, in general, more viscous than cell culture supernatants. Therefore, these samples should be diluted and centrifuged for longer times and at higher speeds, as reported.13 If ultrafiltration is chosen as the enrichment method, undiluted biological samples may clog filter membranes. Also, note that certain commercially available immunoaffinity-based kits for vesicle purification are incompatible with biological samples. Consult manufacturer instructions before using any kit.

    2. What information do you ultimately need to obtain from your vesicle preparation?
    If EVs are being prepared solely to obtain RNA profile or protein cargo information, some degree of damage to the EV membrane (due to shearing forces from repeated, high-g-force ultracentrifugation, for example) might be acceptable. Charge neutralization (“salting out”) generates EV fractions that are suitable for transcriptomics, but not for other analyses, because they are contaminated with non-EV proteins.14 Density gradient ultracentrifugation, when compared to traditional ultracentrifugation and polymeric precipitation, showed the most consistent RNA profiles.9 In one study comparing enrichment of exosome-associated proteins, immunoaffinity capture provided twofold greater enrichment than did ultracentrifugation or density gradients.15

    If the goal is enriching EVs for therapeutic application or to analyze their function, gentler techniques such as tangential flow filtration (TFF) and density gradient ultracentrifugation are more likely to preserve the membrane and, by extension, the biological activity.16

    Exosomes (~30 µm EVs) have been successfully separated from microvesicles (larger, more heterogeneous EVs) using sequential centrifugal ultrafiltration (SCUF) of conditioned media.17 Not only were the protein profiles of the two fractions distinct, but the two fractions also showed differing degrees of invasiveness as measured by a Boyden chamber assay.17

    One factor complicating size-based enrichment of EVs is that vesicles of differing function and biogenesis pathway may overlap in size.18 Because surface markers have, thus far, been a relatively reliable proxy for EV subtype, it is likely that affinity purification approaches hold the greatest promise as an enrichment technique as long as the EVs can be effectively eluted from the solid phase.

    3. Can you tolerate interference from non-EV sample components?
    Non-EV particles may be co-purified with EVs in polymeric precipitation-based techniques.19 Other methods, even if using serum-depleted medium, may yield fractions containing non-EV proteins, such as albumins, lipoproteins, argonaute complexes, or uromodulins. EV samples also contain high amounts of lipids, which can interfere with quantitation of total protein. Proper characterization of such fractions may require assay-free protein analysis, such as by infrared-based quantitation, and bead-based flow cytometric quantitation (Figure 2). Combining protein analysis and relative quantitation can yield the ratio between protein concentration and number of EVs. If this ratio is very high, it may indicate the co-purification of abundant proteins.

    4. How precisely can you control experimental parameters?
    Because vesicles may form as a function of sample collection and preparation, it is important to control the temperature, time, speed, and cell health parameters with which samples are collected and processed. The ISEV published recommended criteria for judging whether a reported EV preparation truly contained functional EVs.2 These criteria called for detailed experimental methods for isolating EVs, including reporting on health and viability of cells of origin.

    Future directions
    Given the heterogeneity of EVs and the subtle differences in their properties, many exciting new technologies are being explored for EV isolation, involving microfluidics, nanointerfaces, electrodialysis, and immunoaffinity capture followed by elution. In addition, methods thought of as “gold standard” today are likely to need further optimization. For example, EVs of diverse physical properties, bearing diverse surface markers, and having diverse mechanisms of biogenesis (Rab-dependent vs. Rab-independent) can only be separated based on kinetics of density gradient flotation because their equilibrium flotation is identical.20 Altering the ultracentrifugation speed can also change the composition of the EV fraction, and refinement of affinity-based approaches may further enable discrimination of EV subtypes.21 Fortunately, close collaborations have emerged among EV researchers and among suppliers of research tools, facilitating coordinated, documented adoption of EV enrichment methods.

 

References
  1. Witwer KW et al. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles. 2013 May 27;2.
  2. Lötvall J et al. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles. 2014 Dec 22;3:26913. doi: 10.3402/jev.v3.26913. eCollection 2014. PubMed PMID: 25536934.
  3. Melo SA et al. Cancer exosomes perform cell-independent microRNA biogenesis and promote tumorigenesis. Cancer Cell. 2014 Nov 10;26(5):707-21.
  4. Shimoda M et al. Loss of the Timp gene family is sufficient for the acquisition of the CAF-like cell state. Nat Cell Biol. 2014 Sep;16(9):889-901.
  5. Webber J, Yeung V, Clayton A. Extracellular vesicles as modulators of the cancer microenvironment. Semin Cell Dev Biol. 2015 Apr;40:27-34.
  6. Chevillet JR et al. Quantitative and stoichiometric analysis of the microRNA content of exosomes. Proc Natl Acad Sci U S A. 2014 Oct 14;111(41):14888-93.
  7. Laurent LC et al. Meeting report: discussions and preliminary findings on extracellular RNA measurement methods from laboratories in the NIH Extracellular RNA Communication Consortium. J Extracell Vesicles. 2015 Aug 28;4:26533.
  8. Webber J et al. Proteomics analysis of cancer exosomes using a novel modified aptamer-based array (SOMAscan™) platform. Mol Cell Proteomics. 2014 Apr;13(4):1050-64.
  9. Van Deun J et al. The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling. J Extracell Vesicles. 2014 Sep 18;3. doi: 10.3402/jev.v3.24858.
  10. Clayton A et al. Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry. J Immunol Methods. 2001 Jan 1;247(1-2):163-74.
  11. Lobb RJ et al. Optimized exosome isolation protocol for cell culture supernatant and human plasma. J Extracell Vesicles. 2015 Jul 17;4:27031.
  12. Yáñez-Mó M et al. Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles. 2015 May 14;4:27066.
  13. Théry C, Amigorena S, Raposo G, Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. 2006 Apr;Chapter 3:Unit 3.22. doi: 10.1002/0471143030.cb0322s30.
  14. Brownlee Z, Lynn KD, Thorpe PE, Schroit AJ. A novel "salting-out" procedure for the isolation of tumor-derived exosomes. J Immunol Methods. 2014 May;407:120-6.
  15. Tauro BJ et al. Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods. 2012 Feb;56(2):293-304.
  16. Lamparski HG et al. Production and characterization of clinical grade exosomes derived from dendritic cells. J Immunol Methods. 2002 Dec 15;270(2):211-26. PubMed PMID: 12379326.
  17. Xu R, Greening DW, Rai A, Ji H, Simpson RJ. Highly-purified exosomes and shed microvesicles isolated from the human colon cancer cell line LIM1863 by sequential centrifugal ultrafiltration are biochemically and functionally distinct. Methods. 2015 Apr 16. pii: S1046-2023(15)00154-1.
  18. Bobrie A, Colombo M, Krumeich S, Raposo G, Théry C. Diverse subpopulations of vesicles secreted by different intracellular mechanisms are present in exosome preparations obtained by differential ultracentrifugation. J Extracell Vesicles. 2012 Apr 16;1.
  19. Zhang B et al. Immunotherapeutic potential of extracellular vesicles. Front Immunol. 2014 Oct 22;5:518. doi: 10.3389/fimmu.2014.00518. doi: 10.3389/fimmu.2014.00518. eCollection 2014. Review. PubMed PMID: 25374570.
  20. Kowal J, Tkach M, Théry C. Biogenesis and secretion of exosomes. Curr Opin Cell Biol. 2014 Aug;29:116-25.
  21. Jeppesen DK et al. Comparative analysis of discrete exosome fractions obtained by differential centrifugation. J Extracell Vesicles. 2014 Nov 6;3:25011. doi: 10.3402/jev.v3.25011. eCollection 2014.

Amedeo Cappione III, Ph.D. (Amedeo.cappione@emdmillipore.com) is a senior scientist and Chandreyee Das, Ph.D., is senior technical writer at MilliporeSigma, the U.S. life science business of Merck KGaA, Darmstadt, Germany.