A novel three step protocol to isolate extracellular vesicles from plasma or cell culture medium with both high yield and purity

Abstract

Extracellular vesicles (EV) are membrane encapsulated nanoparticles that can function in intercellular communication, and their presence in biofluids can be indicative for (patho)physiological conditions. Studies aiming to resolve functionalities of EV or to discover EV-associated biomarkers for disease in liquid biopsies are hampered by limitations of current protocols to isolate EV from biofluids or cell culture medium. EV isolation is complicated by the >10 5-fold numerical excess of other types of particles, including lipoproteins and protein complexes. In addition to persisting contaminants, currently available EV isolation methods may suffer from inefficient EV recovery, bias for EV subtypes, interference with the integrity of EV membranes, and loss of EV functionality. In this study, we established a novel three-step non-selective method to isolate EV from blood or cell culture media with both high yield and purity, resulting in 71% recovery and near to complete elimination of unrelated (lipo)proteins. This EV isolation procedure is independent of illdefined commercial kits, and apart from an ultracentrifuge, does not require specialised expensive equipment.

Skeletal muscle tissue secretes more extracellular vesicles than white adipose tissue and myofibers are a major source ex vivo but not in vivo

“…The findings described here (plasma EVs isolated using precipitation or SEC), by Pua et al (serum EVs isolated using differential ultracentrifugation) (28) and Flaherty et al (ultrafiltration and size exclusion chromatography (7) support two complementary and emerging paradigms. First, the results of these studies are consistent with previous work showing that EVs represent only a small fraction of EV-sized particles found in plasma (12,38). Since cell-specific fluorescent EVs can be detected in abundance ex vivo, our results suggest that EVs secreted by non-vascular cells like SkM myofibers may be largely restricted to specific extracellular compartments.…”

“…The lack of fluorescent EVs in blood plasma (Figure 6) may be explained primarily by coisolated lipoproteins and/or protein aggregates (12,38), but there are at least two other possibilities to be considered. First, EVs secreted from SkM myofibers may be modified as they travel through the interstitial space across the vascular endothelium.…”

A Review of Exosomal Isolation Methods: Is Size Exclusion Chromatography the Best Option?

“…The number of samples that can be processed simultaneously is another limitation associated with use of SEC. Hence, being coupled with another technique [ 108 , 109 ], such as ultracentrifugation, ultrafiltration, or PEG-based precipitation, may be the optimal method of isolation. SEC-coupled techniques generate a high yield of exosomes [ 19 ] that can be used for protein and RNA diagnostics as well as potentially used as a drug or drug delivery system [ 110 , 111 ].…”

Hypoxia and extracellular vesicles: A review on methods, vesicular cargo and functions

“…For complex biofluids, such as plasma, combinations of several isolation methods are being continuously developed to collect EVs of higher purity. Recently, Zhang, Borg, Liaci, Vos, and Stoorvogel (2020) proposed combining PEG polymer precipitation with an iohexol density gradient and then SEC, in that order. A minimal loss of EVs was observed after precipitation, suggesting it as a feasible upstream sample preparation step for inclusion in many EV purification techniques, as an initial concentration step is often required to decrease the sample volume.…”