Comparison of the capillary-channeled polymer (C-CP) fiber spin-down tip approach to traditional methods for the isolation of extracellular vesicles from human urine.

Capillary-channeled polymer fiber (C-CP) solid-phase extraction tips have demonstrated the ability to produce clean and concentrated extracellular vesicle (EV) recoveries from human urine samples in the small EV size range (< 200 nm). An organic modifier-assisted hydrophobic interaction chromatography (HIC) approach is applied in the spin-tip method under non-denaturing conditions-preserving the structure and bioactivity of the recovered vesicles. The C-CP tip method can employ either acetonitrile or glycerol as an elution modifier. The EV recoveries from the C-CP tip method (using both of these solvents) were compared to those obtained using the ultracentrifugation (UC) and polymer precipitation (exoEasy and ExoQuick) EV isolation methods for the same human urine specimen. The biophysical and quantitative characteristics of the recovered EVs using the five isolation methods were assessed based on concentration, size distribution, shape, tetraspanin surface marker protein content, and purity. In comparison to the traditionally used UC method and commercially available polymeric precipitation-based isolation kits, the C-CP tip introduces significant benefits with efficient (< 15 min processing of 12 samples here) and low-cost (< $1 per tip) EV isolations, employing sample volumes (10 µL-1 mL) and concentration (up to 4 × 1012 EVs mL-1) scales relevant for fundamental and clinical analyses. Recoveries of the target vesicles versus matrix proteins were far superior for the tip method versus the other approaches.