Development of a new method for efficiently isolating exosomes from cell culture supernatant
Objective To develop a rapid,simple and efficient method for isolating exosomes from cell culture supernatant,so as to promote the application of exosomes in clinical and disease treatment.Methods A new exosomes extraction system was developed by combining hydroxyl(OH)modified magnetic beads with the polymeric compound Buffer EXD,and the exosomes were extracted from cell culture supernatant by using this system.The expression of exosome marker proteins was detected by Western blot,the particle size and concentration of exosomes were measured by nanoparticles tracking analysis(NTA),and the morphology and structure were determined by transmission electron microscope(TEM).In addition,the extraction methods of exosomes by Buffer EXD system were optimized(pH,Buffer EXD ratio,incubation time,magnetic bead dosage and type),and the effect of exosomes purified by the new system on the proliferation of A549 cells was analyzed by combining cell culture with photomicrograph.Results In exosomes purified by the new system,the expression levels of marker proteins Alix,CD9 and CD81 were high,while the expression level of exosome negative protein Calnexin was low.The exosomes extracted by the new system had a particle size peak of around 100 nm and a classical saucer-like shape.When the new system pH was 5.0,Buffer EXD concentration was 20％,and 1.75％OH magnetic beads were incu-bated with samples for 40 min,the content of marker proteins in exosomes was the highest.Additionally,the exosomes puri-fied by the new system significantly facilitated the proliferation of A549 cells.Conclusion Using OH-modified magnetic beads as the medium,combined with polymeric compound Buffer EXD,a new system was successfully established,which can isolate exosomes with complete structure,obvious morphological characteristics and biological functions from cell culture supernatant rapidly,simply and efficiently.
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