With a growing number of exosome-based delivery vehicles advancing into the clinic for therapeutics and vaccines, more effective isolation and separation strategies are needed. Finnish researchers recently sized up current advances in size-, charge-, and affinity-based strategies for extracellular vesicle isolation and separation, as well as their best uses.
Some of the newest size-based separation techniques include deterministic lateral displacement (DLD) and viscoelastic microfluidic systems. Recent DLD research has focused on “reducing fabrication complexity, clogging, and pressure issues,” Thanaporn Liangsupree, PhD, analytical chemistry researcher at UPM—The Biofore Company; Marja-Liisa Riekkola, PhD, emeritus professor, University of Helsinki; and Evgen Multia, PhD, lecturer, Aalto University, point out.
“For instance,” they note, “thermally-oxidized DLD with tapered shapes has been developed to improve nano-size particle separations. Thermal oxidation was used to obtain nanoscale inter-column spacing and a tapered structure that could separate larger impurities from smaller EV particles.” Combining DLD with dielectrophoretic forces can separate lipoproteins and retroviruses from similarly-sized EVs, they add.
Viscoelastic microfluidic systems, in contrast, use the elastic effects of non-Newtonian media to isolate EVs less than 200 nm. Researchers have combined elastic and inertial lift to isolate EVs from cell culture and human serum.
Other newer methods separate EVs based on their hydrophobic interaction. Solid-phase extraction using a capillary-channeled polymer fiber to isolate EVs from cell cultures, urine, and blood plasma is one example. And, when combined with hydrophobic interaction chromatography (HIC), EVs have been separated from lipoproteins. Multimode chromatography is also proving successful.
Size exclusion chromatography (SEC) is the most frequently mentioned technique for EV isolation. It excels at separating large biomacromolecules—most recently from fungal and plant sources—in addition to human and animal samples.
When isolating EVs from blood-derived samples, however, SEC is challenged by the co-elution of similarly-sized lipoprotein contaminants. Doubling the pore size from 35 nm to 70 nm increased ratios of EV to apolipoprotein (Apo) A by 34-fold, ApoB by 44-fold, and proteins 120-fold, Liangsupree and colleagues note, referencing a 2024 paper by research associate Jillian W.P. Bracht, PhD, et al. of the University of Amsterdam.
While cross-linked agarose resins typically are used for EV separation by SEC, hydrophilic porous silica gel (pore size: 73 nm) successfully separated EVs from urine. Able to withstand higher pressure and flow rates, this method isolated EVs at a rate of 2 mL per minute with an approximate yield of 77.5%. Combining SEC with anion exchange chromatography “improves proteomic analysis for plasma-derived EVs,” they noted, citing Canadian research.
Filtration is another popular, scalable option, and ultrafiltration—including tangential flow filtration—is particularly attractive for small-volume EV enrichment. The team evaluated nearly 30 techniques, including recent flow field-flow fractionation-based applications, noting samples, separation systems, applied pore sizes, and references.
Affinity and immunoaffinity-based EV isolation is also popular. “Recent studies have covered a wider range of disease- and tissue-specific immunoaffinity ligands for targeted EV isolation,” they point out. Recent work has used antibodies against aspartate β-hydroxylase to isolate ASPH+ EVs and thus diagnose lymph node metastasis, and human epidermal growth factor receptor 2 (HER2) to monitor and diagnose breast cancer.
“Based on the published studies, it is evident that only combined techniques and approaches can meet a high number of criteria to be beneficial in diagnostics and therapeutics,” they report. Each technique has great potential, but also clear concerns, leading the team to conclude that combining techniques is the most useful approach.
