Isolation and analysis methods of extracellular vesicles (EVs)

Abstract

Extracellular vesicles (EVs) have been recognized as an evolving biomarker within the liquid biopsy family. While carrying both host cell proteins and different types of RNAs, EVs are also present in sufficient quantities in biological samples to be tested using many molecular analysis platforms to interrogate their content. However, because EVs in biological samples are comprised of both disease and non-disease related EVs, enrichment is often required to remove potential interferences from the downstream molecular assay. Most benchtop isolation/enrichment methods require > milliliter levels of sample and can cause varying degrees of damage to the EVs. In addition, some of the common EV benchtop isolation methods do not sort the diseased from the non-diseased related EVs. Simultaneously, the detection of the overall concentration and size distribution of the EVs is highly dependent on techniques such as electron microscopy and Nanoparticle Tracking Analysis, which can include unexpected variations and biases as well as complexity in the analysis. This review discusses the importance of EVs as a biomarker secured from a liquid biopsy and covers some of the traditional and non-traditional, including microfluidics and resistive pulse sensing, technologies for EV isolation and detection, respectively.

Keywords: Extracellular vesicles; microfluidics; molecular cargo; nanoparticle tracking analysis; resistive pulse sensing.

Figure 1

(A) Sub-types of extracellular vesicles including microvesicles, exosomes, and apoptotic bodies. (B) Size ranges of the three sub-types of extracellular vesicles of which exosomes are the smallest with a range from 30 to 150 nm. Microvesicles range from 100 to 1000 nm in size, but the size ranges from 100 to 400 nm when they are present in the circulatory system. Apoptotic bodies range from 1 µm up to 5 µm in size (Reproduced from^[10]).

Figure 2

Exosomes are small EVs (sEVs) with the size range from 30 to 150 nm. Exosomes carry various types of molecules originating from the cell-of-origin including proteins, nucleic acids, lipids, and metabolites. Exosomes also play essential roles in cellular communication and regulation (Reproduced from^[20]).

Figure 3

Conventional methods for EV enrichment. (A) Polymer-based enrichment: Precipitation with polyethylene glycol (PEG) (reproduced from Reference 100). (B) Filtration and ultrafiltration for EV isolation: normal prefiltration can collect sEVs and particles into the bottom layer of the culture dish. The bottom layer needs to be processed through tangential ultrafiltration, and the retentate is collected. Further ultrafiltration with expected pore size can be further processed and the EVs with a size smaller than the pore size will be present in the permeate (reproduced from^[28]). (C) Ultracentrifugation for EV isolation (Reproduced from^[111]). (D) Summary of yield and purity of sEVs isolated by SEC or UC: Normalization of APOB signal to CD81 content as an estimate of sEV purity from lipoproteins, also demonstrated almost 60 times higher APOB/CD81 ratio in the peak sEV fraction of SEC (5.5 ml) compared to the UC samples. SEC resulted in a higher yield of sEVs but with marked contamination by soluble protein and lipoproteins (reproduced from^[116]). (E) Analysis of EVs by NTA demonstrates differences in size distribution. Black bars indicate the absolute number of vesicles isolated from 1 ml of serum; red diamonds plotted against the right x-axis represent vesicle purity defined as the particle to protein ratio. While precipitation most efficiently isolated EVs from serum, SEC-based isolation yielded fewer but more pure vesicles. Asterisks indicate significant differences in particle numbers compared to miRCURY. *P < 0.05; **P < 0.01; NS: not significant. All data are mean ±SD for five volunteers and five sepsis patients (reproduced from^[117]). NTA: Nanoparticle tracking analysis.

Figure 4

(A) ^newExoChip design, which features 30 × 60 circular patterns with a diameter of 500 μm in standard glass microscope slides. The mechanism of capture and release of cancer-associated exosomes using Ca²⁺-dependent binding between PS and annexin V and ethylenediaminetetraacetic acid (EDTA)-based Ca²⁺ chelation. The micrograph shows capture and released exosomes (reproduced from^[131]). (B) Nano-interfaced microfluidic exosome platform (nano-IMEX). Schematic of a single-channel PDMS/glass device with expanded-view highlighting the coated PDMS chip containing an array of Y-shaped microposts. The surface of the channel and microposts coated with graphene oxide (GO) and polydopamine (PDA) as a nanostructured interface for the sandwich ELISA with fluorescence signal amplification (reproduced from^[123]). (C) 3D herringbone nanopatterns are designed on a microfluidic device with the ability to detect tumor-associated EVs in plasma with a minimum of 200 vesicles per 20 µL. The nano-structures were used to increase the surface area, content mass transfer, and EV capturing speed, and reduce the hydrodynamic resistance (reproduced from^[129]). (D) Microfluidic device made from cyclic olefin polymer (COP), which allows for high-rate production at a low cost to accommodate diagnostic applications. CAD drawing of a 7-bed EV Microfluidic Affinity Purification (EV-MAP) showing the distribution channels and the diamond-shaped micropillars of the device. NTA and TEM images of EVs isolated from a clinical sample by PEG precipitation and affinity selected with anti-CD8 mAb using the EV-MAP device. Heat map analysis of clinical samples (marked with numbers) and healthy donor for 5 genes whose up-regulation is associated with acute ischemic stroke (reproduced from^[139]). (E) ExoDisc integrated system that combines a sequential filtration and centrifugation steps used for low viscosity fluids. The EVs are collected between filter I and filter II. The filters can be replaced with different pore sizes for different expected size range selection (reproduced from^[33]).

Figure 5

The principle of NTA measurements and the Stokes-Einstein equation for the analysis of particle size (reproduced from^[145]).

Figure 6

Concentration variations for different types of samples including EVs from PC-3 cell culture media, EVs from Jurkat cell culture media, Outer membrane vesicle from Neisseria meningitidis, microvesicles from monocytes, article vesicles, polystyrene latex beads (100 nm), and silica microspheres (150 nm). The samples were tested on 6 different days and the variation is from 1% to 18% (Reproduced from^[143]).

Figure 7

(A) Transmission electron microscopy image of EVs (scale bar = 100 nm). (B) Scanning electron microscope image of EVs showing the circular shape of the EVs (reproduced from^[151]). (C) Scanning electron microscope image of EVs, which shows cup-shaped EVs (reproduced from^[150]). (D) Atomic force microscope image for EVs (reproduced from^[156]).

Figure 8

(A) The principle of flow cytometry. (B) A flow cytometry platform designed by Friedrich et al.^[164] The nanofluidic device contained 100 nanochannels with a width of 300 nm and the dye-labeled EVs could be sensed and recorded by a fluorescent microscope (reproduced from^[164]).

Figure 9

The evolution of resistive pulse sensing (RPS) from fixed pore with micro-scale to flexible pore with micro- to nano-scale sizes. RPS can also be applicable as a Coulter counter for EV analysis with the proper sized pore (reproduced from^[171]).

Figure 10

(A) The principle of RPS measurements with constant potential or current clamping across the measuring pore. The amplitude profile shows a relationship with particle size. The particle size distribution information can be determined by analyzing the event amplitude, and the event frequency can be analyzed for concentration information (reproduced from^[172]). (B) The RPS is also used to study the particle shape, movement, and interaction with the solvent. The event can express the particle shape and also the rotation level and axis (Reproduced from^[183]).