Abstract
Extracellular vesicles (EVs) are membrane-bound vesicles released by cells and act as media for transfer of proteins, small RNAs and mRNAs to distant sites. They can be isolated by different methods. However, the biological activities of the purified EVs have seldom been studied. In this study, we compared the use of ultracentrifugation (UC), ultra-filtration (UF), polymer-based precipitation (PBP), and PBP with size-based purification (PBP+SP) for isolation of EVs from human endometrial cells and mouse uterine luminal fluid (ULF). Electron microscopy revealed that the diameters of the isolated EVs were similar among the tested methods. UF recovered the highest number of EVs followed by PBP, while UC and PBP+SP were significantly less efficient (P<0.05). Based on the number of EVs-to-protein ratios, PBP had the least protein contamination, significantly better than the other methods (P<0.05). All the isolated EVs expressed exosome-enriched proteins CD63, TSG101 and HSP70. Incubation of the trophoblast JEG-3 cells with an equal amount of the fluorescence-labelled EVs isolated by the studied methods showed that many of the PBP-EVs treated cells were fluorescence positive but only a few cells were labelled in the UC- and UF-EVs treated groups. Moreover, the PBP-EVs could transfer significantly more miRNA to the recipient cells than the other 3 methods (P<0.05). The PBP method could isolate EVs from mouse ULF; the diameter of the isolated EVs was 62±19 nm and expressed CD63, TSG101 and HSP70 proteins. In conclusion, PBP could best preserve the activities of the isolated EVs among the 4 methods studied and was able to isolate EVs from a small volume of sample. The simple setup and low equipment demands makes PBP the most suitable method for rapid EV assessment and isolation of EVs in clinical and basic research settings.
Highlights
Extracellular vesicles (EVs) are nanometer-sized membrane-bound vesicles secreted by most living cells into their extracellular environment
There was no significant difference in size between the EVs isolated by the 4 different methods (Fig 1E, UC: 54.04±17.03 nm, polymer-based precipitation (PBP): 70.71±35.16 nm, PBP with size-based purification (PBP+SP): 60.43±45.40 nm and UF: 53.30 ±27.58 nm)
The results were expressed as mean number of EVs and showed a pattern similar to that obtained by Electronic microscopy (EM), i.e. UF has the highest recovery while PBP+SP has the least number of EVs (Fig 1G: PBP: 4.0±0.9, PBP+SP: 0.7±0.5, UF: 122.9 ±9.0 and UC: 2.8±3.3)
Summary
Extracellular vesicles (EVs) are nanometer-sized membrane-bound vesicles secreted by most living cells into their extracellular environment. They are subcellular components enclosed in a membrane with a lipid bilayer. Microvesicles and apoptotic bodies are different types of EVs. Exosomes are typically of sizes 30–100 nm in diameter [1]. Exosomes are typically of sizes 30–100 nm in diameter [1] They are formed within cells by inward budding of late endosomes, called multi-vesicular bodies. The exosomes are released into the extracellular environment when the multi-vesicular bodies fuse with the plasma membrane [2,3]. Different from the exosomes, microvesicles bud directly from the plasma membrane, and are usually 50–1000 nm in diameter. Given the overlapping size ranges, it is well accepted that EV preparations are generally heterogeneous [8,9]
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