Study On Polymer-induced Magnetic Beads Coprecipitation Method For Isolation And Purification Of Exosomes

In order to clearly reveal the function and mechanism of action of exosomes in life phenomena and to use them as markers for diagnosis and as a carrier for treatment,it is necessary to obtain exosomes with high purity and sufficient quantity,but to date,exosomes efficient separation and purification technologies remain a challenge.The gold standard ultracentrifugation separation method relies on large instruments,which has low throughput and time-consuming labor.However,although the number of exosomes separated by specific reagents is large,the purity is low,which has a great influence on downstream applications.Although the separation method of the flow channel is high in purity,but the yield of exosome is low,and it is difficult to provide a sufficient amount of exosomes for subsequent application or analysis.Therefore,this project focuses on the development of an efficient and simple exosomal separation method that can achieve high purity and high yield,in order to meet the requirements of exosomes for downstream RNA and protein detection and as a novel biological carrier.This study proposed a novel separation and purification method based on magnetic microsphere-mediated polymer coprecipitation of exosomes.First,based on the action of the hydrophilic polymer PEG,exosomes are induced to deposit on the surface of the magnetic beads,and the magnetic beads are captured by simple magnetic separation,followed by elution by PBS to obtain an exosomal dispersion.Secondly,the effects of PEG concentration,separation and purification strategy,magnetic microsphere dosage,etc.on the separation and purification performance of exosomes and the basic rules of separation of exosomes were studied systematically.Western blotting was used to examine the morphology,particle size,concentration,and specific markers on the surface of the exosomes,and to evaluate the purity and yield performance of the established exosomes.Finally,the established separation and purification method based on magnetic microsphere-mediated polymer coprecipitation exosomes was applied to two typical samples of real plasma and cell culture fluid,and the optimal conditions for plasma and cell culture fluid were proposed respectively.The separation and purification strategy was compared with the gold standard for ultracentrifugation of exosomes.Studies have shown that the exosome product extracted by the MB-PEG method proposed in this study has a purity of 10¹⁰-10¹² particles per milligram of heteroprotein,and the particles recovery rate is close to 65%-80%.Both RNA and protein content are higher than the ultracentrifugation method.The novel exosomes isolated and purified in this study have been used to separate and purify exosomes.Finally,the high-efficiency separation of exosomes in plasma and cell culture fluids is realized.It has simple operation,large sample separation flux,short time-consuming and low cost.And the obtained exosome product is intact in morphology,retaining abundant inclusions,and the purity and yield can meet the downstream application.