Quantification of Small Extracellular Vesicles by Size Exclusion Chromatography with Fluorescence Detection

Chemical analysis of small extracellular vesicles (sEVs) circulating in body fluids holds potentials in noninvasive diagnosis of diseases and evaluation of therapeutic treatments. However, quantification of sEVs remains a challenge due to lacking of cost-effective analytical protocols. Herein we report a facile method based on size exclusion chromatography with fluorescence detection (SEC-FD) for sEVs quantification. After removal of cells and cell debris, a 0.50 mL sample (e.g., cell culture medium) is incubated with CM-Dil dye to fluorescently label sEVs. The incubation solution is then separated on a SEC column packed with Sepharose CL-4B. The eluent is monitored fluorescently at Ex553 nm/Em570 nm by using a fluorometer equipped with a 50-mu L flow through cuvette. Separation efficiency of the proposed SEC-FD method was evaluated by analyzing 100 nm liposomes and albumin-FITC conjugate. Liposomes were eluted out in less than 6 min, about 10 min before albumin-FITC. A separation repeatability (RSD in retention time) of 1.4% (n = 5) was obtained for liposomes. In analysis of cell culture media, linear calibration curves based on SEC-FD peak height versus sEVs concentration were obtained with r(2) value of 0.996. Intraday quantification repeatability (RSD in peak height) was 3.2% (n = 5). The detection limit was estimated to be 2.9 X 107 exosome particles/mL. The proposed assay was applied to the first study of sEVs secretion from TK6 cells cultured in serum-free medium for a culturing period from 1 to 48 h.