A New Method For Enrichment Of Extracellular Vesicles

Objective:Extracellular vesicles（EVs）are nanoscale vesicles secreted by various types of cells into the extracellular.EVs play pivotal roles in physiological and pathophysiological processes such as waste removal and cell-to-cell communication,etc.Compared to EVs obtained from body fluids or cell culture supernatants,EVs isolated directly from tissues possess a number of advantages,the main features are tissue specificity and accurate characterization of the tissue microenvironment,etc.In addition to this,unlike 2D（two-dimensional）cell culture,3D（three-dimensional）cell culture is thought to be more capable of mimicking in vivo cell behavior,and 3D culture-derived EVs have been shown to be more similar to EVs obtained from patients.Thus.Tissue-derived EVs（Ti-EVs）and 3D cultured cell-secreted EVs are gradually gaining widespread attention.Ti‐EVs are present in the interstitium of tissues and play pivotal roles in intercellular communication.Ti‐EVs may be used to gain insights into the development and progression of cancer.Most EVs studies to date have been conducted on somatic fluids or 2D cultured cells.Due to technical limitations,the number of studies on Ti‐EVs and on EVs secreted by 3D cultured cells remained limited.Therefore,this study establishes a method to isolate EVs directly from tissues and from cells in 3D culture.Method:First,the tissue is subjected to a mild enzymatic treatment,as well as filtration and low-speed centrifugation to remove cell debris,dead cells,etc.We extracted EVs by combining the kit with Ti O₂ microsphere material.The crude separation of EVs was first performed using the EVs extraction and purification kit,and then enriching the tissue EVs by coordination binding between Ti O₂ and phosphate groups on the surface of phospholipid bilayers.The purity of EVs was further improved.The particle size distribution,particle number concentration and morphological characteristics of EVs were examined using BCA,NTA and TEM.Identification of signature proteins on the surface of EVs by Western blot.It was subsequently applied to the extraction of EVs from liver cancer tissues and proteomic analysis;（2）We built 3D tumor cell spherical structures and synthesize magnetic nanomaterials to isolate EVs from 3D tumor cells.The synthesized materials were characterized using TEM,XPS and XRD experiments.And the differences of EVs extracted from 3D and 2D cultured cells were compared by TEM,NTA,Western blot experiments and mass spectrometry data.Results:（1）After protein quantification（BCA）,nanoparticle tracking analysis（NTA）experiments confirmed that the method was able to extract high-quality EVs.TEM showing the complete structure of EVs.Western blot confirmed the presence of EV signature proteins TSG101,Syntenin-1,CD63,and no cellular debris signature proteins Calnexin,VDAC,GM130.The results indicated that 20 mg Ti O₂ microspheres capturing EVs in 200μL of tissue solution for 10 min was the optimal condition.In addition,LC-MS results showed that a total of 1966 EVs proteins were identified by the kit-Ti O₂ method,more than that obtained by ultracentrifugation,using the kit or the Ti O₂ method alone.The method was applied to the extraction of EVs from liver tissues of healthy mice and HCC mice.A total of 70 differential proteins were identified in the two sets of samples by proteomic analysis.Twenty-five of these proteins were upregulated in EV of HCC mouse liver tissues,and 11 of them have been reported to be closely associated with HCC development.（2）Due to the small specific surface area of micrometer sized Ti O₂spheres and their limited enrichment efficiency,we also synthesized a magnetic nanomaterial Fe₃O₄@Ti O₂ to enrich EVs.TEM,XPS and XRD were used to characterize the material,and the results showed that we successfully synthesized nanomaterials.We also successfully established 3D tumor cell spherical structures and used the magnetic nanomaterial Fe₃O₄@Ti O₂ to extract EVs from 3D cultured cells.We further compared the EVs extracted from 3D cultured cells with those from 2D cultured cells by TEM,NTA and Western blot experiments.We found that the efficiency of EV secretion from 3D cultured cells was significantly higher than that from 2D cultured cells.Further application of the material for EVs extraction from 3D and2D cultured cells and proteomics analysis,and 1068 and 1022 proteins were identified,respectively.We screened 51 differential proteins,of which 28 were up-regulated in 3D-EVs and 23 were down-regulated in 3D-EVs.Conclusion:（1）We developed a simple method for direct enrichment of EVs from tissues.The extracted EVs were characterized by using NTA,BCA,TEM and Western blot methods.The results show that our method is able to extract EVs in higher quantities and with higher purity.Proteomics analysis showed that the method was able to identify up to1966 EV proteins with high sensitivity and reproducibility.This method was subsequently applied to liver tissues of hepatocellular carcinoma mice and healthy mice for EVs differential protein screening.（2）We synthesized a magnetic nanomaterial Fe₃O₄@Ti O₂for enrichment of EVs in 3D culture cell.The material has the advantages of superparamagnetism,high specific surface area,easy separation.The method has the advantages of simplicity,rapidity,high sensitivity and high enrichment efficiency.We compared the EVs extracted from cells cultured in 2D and 3D systems by NTA,Western blot experiments and proteomic analysis.The results showed that 3D cultured cells secreted EVs significantly more efficiently than 2D cultured cells.And there are some differences between the two EVs proteins.