Establishment Of Dichotomic Size-exclusion Chromatography In Serum Extracellular Vesicles Separation And Prilimenary Application Demostration

Extracellular vesicles(EVs)are particles with bimolecular phospholipids that are secreted naturally by cells and have no ability to replicate.Numerous studies have shown that EVs play an important role in disease diagnosis,prognosis and treatment.We previously developed a dichotomic SEC method to allow rapid EV isolation from FBS quickly.In this study,we further used human serum(HS)to optimize this SEC method in terms of Sepharose CL resin selections and the bed volume determination.Among all conditions tested in this study,the 20 m L CL-6B SEC column had the best resolution of 1.83 in the separation of EVs from proteins.With the 20 m L CL-6B SEC column,we could harvest(1.32±0.5)×10¹²EVs particles from 1 m L HS,with the particle/protein ration of(1.2±0.9)×10⁹ particles/?g protein.Such a purity was superior to the performance of the q EV kit.According to the immunblotting analysis results,the EVs fraction acquired from this dichotomic SEC showed clear integrin and sytenin bands,while the contaminants of albumin and lipoproteins were largely removed.Nanoparticle tracking analysis(NTA)results also showed that the EV particles were within the size range of 50-200 nm in diameter,which was comparable with the TEM observations.Regarding the application demonstration,we used dichotomic SEC method to separate EVs from CRC patient,and found that miR-21 was largely enriched in the protein fraction,but not in the EV fraction.HBV miR-3 was the first autocoded micro RNA of Hepatitis B Virus found in plasma EVs,and its content was highly positively correlated with HBs Ag in peripheral blood EVs of patients with chronic Hepatitis B(CHB).In our prvious study,in the peripheral blood EVs of CRC patients,the quantity of HBV miR-3 was supplementary to pg RNA to estimate the transcription activity of HBV reservoir.We isolated EVs from the supernatants of Huh-7 cells over-expressing HBV miR-3.We found that HBV miR-3 was again primarily enriched in the protein fraction,but not in the EV fraction.In conclusion,we established a dichotomic SEC method that could rapidly isolate EVs from HS potentially applicable to clinical use.Furthermore,with this SEC method,we found that miR-21 and HBV miR-3 were enriched in the protein fraction rather than in EVs.