Asymmetric-flow field-flow fractionation technology for exomere and small extracellular vesicle separation and characterization


We describe the protocol development and optimization of asymmetric-flow field-flow fractionation (AF4) technology for separating and characterizing extracellular nanoparticles (ENPs), particularly small extracellular vesicles (sEVs), known as exosomes, and even smaller novel nanoparticles, known as exomeres. This technique fractionates ENPs on the basis of hydrodynamic size and demonstrates a unique capability to separate nanoparticles with sizes ranging from a few nanometers to an undefined level of micrometers. ENPs are resolved by two perpendicular flows—channel flow and cross-flow—in a thin, flat channel with a semi-permissive bottom wall membrane. The AF4 separation method offers several advantages over other isolation methods for ENP analysis, including being label-free, gentle, rapid (<1 h) and highly reproducible, as well as providing efficient recovery of analytes. Most importantly, in contrast to other available techniques, AF4 can separate ENPs at high resolution (1 nm) and provide a large dynamic range of size-based separation. In conjunction with real-time monitors, such as UV absorbance and dynamic light scattering (DLS), and an array of post-separation characterizations, AF4 facilitates the successful separation of distinct subsets of exosomes and the identification of exomeres. Although the whole procedure of cell culture and ENP isolation from the conditioned medium by ultracentrifugation (UC) can take ~3 d, the AF4 fractionation step takes only 1 h. Users of this technology will require expertise in the working principle of AF4 to operate and customize protocol applications. AF4 can contribute to the development of high-quality, exosome- and exomere-based molecular diagnostics and therapeutics.

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Fig. 1: Schematic illustration of the AF4 working principle.
Fig. 2: Influence of cross-flow on AF4 fractionation.
Fig. 3: Effect of the channel height upon AF4 fractionation.
Fig. 4: Effect of the focus time upon AF4 fractionation.
Fig. 5: Comparison of the AF4 performance for separating EVs using different membranes.
Fig. 6: Examination of the sample (B16-F10 sEVs) loading capacity for AF4 analysis.
Fig. 7: Schematic illustration of the overall procedure, the instrument flow route and operative methods for AF4 of ENPs.
Fig. 8: Representative AF4 fractionation analysis of B16-F10 sEVs.

Data availability

All Astra 6 data files used for producing the plots presented in figures have been deposited at


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We are grateful for the great AF4 technical support from Wyatt Technology. We thank our colleagues I. Matei and C. Kenific for comments on this protocol. Our study was supported by the National Cancer Institute (U01-CA169538, D.L.), the National Institutes of Health (R01-CA169416, D.L.; R01-CA218513, D.L. and H.Z.), the United States Department of Defense (W81XWH-13-1-0249, D.L.; W81XWH-13-1-0427, D.L.), the Sohn Conference Foundation (H.Z.), the Children’s Cancer and Blood Foundation (D.L.), the Manning Foundation (D.L.), the Hartwell Foundation (D.L.), the Nancy C. and Daniel P. Paduano Foundation (D.L.), The Starr Cancer Consortium (D.L. and H.Z.), the Pediatric Oncology Experimental Therapeutic Investigator Consortium (POETIC, D.L.), the James Paduano Foundation (D.L.), the National Institutes of Health/WCM CTSC (NIH/NCATS UL1TR00457 (H.Z.); NIH/NCATS UL1TR002384 (D.L. and H.Z.)), Thompson Family Foundation (D.L.), and Malcolm Hewitt Wiener Foundation (D.L.).

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D.L. and H.Z. designed and technically developed the protocol. H.Z. performed the experiments. D.L. and H.Z. analyzed the data and wrote the manuscript.

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Correspondence to Haiying Zhang or David Lyden.

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Journal peer review information: Nature Protocols thanks Pieter Vader and other (anonymous) reviewer(s) for their contribution to the peer review of this work.

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Zhang, H. et al. Nat. Cell Biol. 20, 332–343 (2018):

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Supplementary Figure 1 Estimation of the yield recovered for exomere, Exo-S and Exo-L derived from B16-F10 and AsPC-1.

Shown in a and b are the yield recovered for exomere, Exo-S and Exo-L derived from B16-F10 and AsPC-1 (100 μg of sEV input for AF4), respectively. c and d show the average concentration of unconcentrated fraction post AF4 fractionation for exomere, Exo-S and Exo-L derived from B16-F10 and AsPC-1, respectively. Data are presented as mean ± s.e.m. and three independent experiments for each cell type are analyzed.

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Zhang, H., Lyden, D. Asymmetric-flow field-flow fractionation technology for exomere and small extracellular vesicle separation and characterization. Nat Protoc 14, 1027–1053 (2019).

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