Characterization of Human Glioblastoma versus Normal Plasma-Derived Extracellular Vesicles Preisolated by Differential Centrifugation Using Cyclical Electrical Field-Flow Fractionation

Abstract

Although many properties for small extracellular vesicles (sEVs, formerly termed “exosomes”) isolated at ∼100 000g are known, a wide range of values are reported for their electrophoretic mobility (EM) measurements. This paper reports for the first time the effect of dilution on the EM of U87 glioblastoma cell-derived and plasma-derived sEVs and medium size EVs (mEVs, commonly termed “oncosomes”) preisolated by differential centrifugation. Furthermore, the effect of resalting on the EM of sEVs and mEVs was evaluated. The EM of U87 sEVs and U87 mEVs showed an increase as the salt concentration decreased to 0.005% of the initial salt concentration. However, for the plasma sEVs and plasma mEVs, the electrophoretic mobility increased as the salt concentration decreased to 0.01% of the initial salt concentration and then increased to its initial value when the salt concentration decreased to 0.005% of the initial salt concentration. For both U87 and plasma sEVs and mEVs, the EM remained almost constant when the concentration of the particles changed and the salt concentration was kept the same as its initial value. This indicates that the EM of EVs is only a function of the salt concentration of the buffer and is independent of the concentration of the particles. The sEVs and mEVs were separated with cyclical ElFFF for the first time. The results indicate that ElFFF was able to fractionate the EVs, and a crescent-shaped trend was found for the retention time when the applied AC voltage was altered (increased).

Development and Testing of a Continuous Flow-Electrical-Split-Flow Lateral Transport Thin Separation System (Fl-El-SPLITT)

“…Second, the electrophoretic mobilities of sEVs and mEVs were close to each other, which consequently, led to their migration toward the top electrode at the same rate. This possibility is supported by our previous cyclical electrical field-flow fractionation findings demonstrating subtle differences between the electrophoretic mobilities of sEVs and mEVs …”

“…This possibility is supported by our previous cyclical electrical field-flow fractionation findings demonstrating subtle differences between the electrophoretic mobilities of sEVs and mEVs. 23 Comparing the results obtained from SA14 and SA15 (in which the cross flow was applied) with the ones obtained from the Tasci device (no cross flow) indicates that applying cross flow improved the separation effectiveness. This is due to the fact that applying cross flow increased the differentiation in the movement of large particles from the small ones by applying greater opposing hydrodynamic force on the large particles than the small ones.…”

“…sEVs and mEVs were purified from the supernatants of a cultured U87 glioma cancer cell line using standard ultracentrifugal techniques at The University of Louisville. Briefly, mEVs were isolated post 10,000 g and sEVs isolated post 110,000 g according to established methods . After the isolating process, fluorescent DiIC 18 (3) (DiI) and DiOC 18 (3) (DiO) dyes were, respectively, used to dye sEVs and mEVs.…”

“…Briefly, mEVs were isolated post 10,000g and sEVs isolated post 110,000g according to established methods. 23 After the isolating process, fluorescent DiIC 18 (3) (DiI) and DiOC 18 (3) (DiO) dyes were, respectively, used to dye sEVs and mEVs. Samples were frozen, shipped overnight, and stored at −80 °C until they were used.…”

Recent Advances in Extracellular Vesicles as Drug Delivery Systems and Their Potential in Precision Medicine

“…Cancer EVs can be isolated from fluids such as plasma [ 27 , 28 , 29 ]. For prostate cancer [ 30 ] and bladder cancer [ 31 ] urine may also constitute a source of vesicles.…”