Size characterization and quantification of exosomes by asymmetrical-flow field-flow fractionation.
In the past few years extracellular vesicles called exosomes have gained huge interest of scientific community since they show a great potential for human diagnostic and therapeutic applications. However, an ongoing challenge is accurate size characterization and quantification of exosomes because of the lack of reliable characterization techniques. In this work, the emphasis was focused on a method development to size-separate, characterize, and quantify small amounts of exosomes by asymmetrical-flow field-flow fractionation (AF4) technique coupled to a multidetection system (UV and MALS). Batch DLS (dynamic light-scattering) and NTA (nanoparticle tracking analysis) analyses of unfractionated exosomes were also conducted to evaluate their shape and internal structure, as well as their number density. The results show significant influence of cross-flow conditions and channel thickness on fractionation quality of exosomes, whereas the focusing time has less impact. The AF4/UV-MALS and DLS results display the presence of two particles subpopulations, that is, the larger exosomes and the smaller vesicle-like particles, which coeluted in AF4 together with impurities in early eluting peak. Compared to DLS and AF4-MALS results, NTA somewhat overestimates the size and the number density for larger exosome population, but it discriminates the smaller particle population.
National Institute of Chemistry , Laboratory for Polymer Chemistry and Technology, Hajdrihova 19, 1000 Ljubljana, Slovenia.
Simona
S
Sitar
University of Ljubljana , Faculty of Medicine, Institute of Biochemistry, Vrazov Trg 2, 1000 Ljubljana, Slovenia.
Anja
A
Kejžar
National Institute of Chemistry , Laboratory for Polymer Chemistry and Technology, Hajdrihova 19, 1000 Ljubljana, Slovenia.
David
D
Pahovnik
University of Ljubljana , Faculty of Chemistry and Chemical Technology, Department of Chemistry and Biochemistry, Večna pot 113, 1000 Ljubljana, Slovenia.
Ksenija
K
Kogej
National Institute of Biology , Večna pot 111, 1000 Ljubljana, Slovenia.
Magda
M
Tušek-Žnidarič
University of Ljubljana , Faculty of Medicine, Institute of Biochemistry, Vrazov Trg 2, 1000 Ljubljana, Slovenia.
Metka
M
Lenassi
National Institute of Chemistry , Laboratory for Polymer Chemistry and Technology, Hajdrihova 19, 1000 Ljubljana, Slovenia.
Ema
E
Žagar
D002460:Cell Line; D055354:Exosomes / Q000737:chemistry*; D031521:Fractionation, Field Flow / Q000379:methods*; D006801:Humans; D053758:Nanoparticles / Q000032:analysis; D013997:Time Factors