Researchers from Weill Cornell Medicine 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.
Schematic illustration of the AF4 working principle
a-c, Side views of the AF4 channel, whose height is usually several hundreds of micrometers. The part sizes shown in the figure are for illustration only and not drawn to scale. a, In the focus stage, two flows in opposing directions (fine blue arrows) are pumped into the channel from the inlet and outlet ports and balanced near the injection port. Samples are injected during the focus stage and focused in a thin band by the two opposing flows. Particles reach heights related to their diffusion coefficients. b, In the elution stage of the normal mode, a single direction of channel flow from the inlet to the outlet is applied, and particles with small hydrodynamic size and high diffusion coefficient are eluted at an early time point, whereas particles with large hydrodynamic size and low diffusion coefficient elute later. c, When the physical size of a particle is too large, as compared to the channel height, to be considered as a point mass, it elutes in the steric mode. In contrast to the normal mode shown in b, in steric mode large particles elute earlier than the smaller ones. The fine blue arrows indicate the channel flow (horizontal arrows) and cross-flow (vertical arrows); the fine black arrows illustrate the channel flow carrying the ENPs through the channel during the elution step (the lengths of the arrows represent the magnitude of the flow rate); the thick blue arrows represent the inlet and outlet flows; and the red-outlined arrows depict the injection flow (the filled arrow indicates the injection flow is ON, and the empty arrows indicate the injection flow is OFF). All the arrowheads point in the flow direction. The fine green arrows indicate the direction of particle movement caused by the cross-flow; the fine red arrows in b and c indicate the direction of particle movement due to their diffusion (b) or the physical position of the particles in the channel due to their physical sizes (c). Rh, hydrodynamic radius.