Extraction Of Small Extracellular Vesicles From Human Liver Cancer Tissue Based On A New Enrichment Method

Small extracellular vesicles(sEVs)are of great value in the diagnosis and treatment of diseases.The sEVs obtained by appropriate procedures of isolation and purification directly from various tissues can reflect the physiological and pathological status of the organism.However,the quality of sEVs during isolation is affected by many factors,including contamination of tissues and cellular debris,the type of tissue digestive enzymes,and the storage status of tissues.In the present study,we established a method for the isolation and purification of sEVs derived from liver cancer tissues in different storage states,including tissue-derived sEVs(tdsEVs)and cultured explantsderived sEVs(cedsEVs),by comparing the quality of sEVs obtained from liver cancer tissues with different concentrations of digestive enzymes and incubation times.Nanoflow cytometry(Nano FCM)showed that the tdsEVs obtained by our established method(method 3,an improved method for differential ultracentrifugation)were of higher purity than those obtained by differential ultracentrifugation.Thus,our study established a simple and effective method for the isolation of high-quality sEVs and determined the optimal experimental conditions for the isolation and enrichment of sEVs from liver cancer tissues.These results will provide important data and methodology to support the study of tissue sEVs.Objective:1.To explore the enrichment methods for high quality sEVs.2.To determine the optimal conditions for the isolation of sEVs from human liver cancer tissues.3.To investigate whether tdsEVs extracted by method 3(modified differential ultracentrifugation)are biologically active.Methods:1.Particle concentration,protein content and purity analysis of tdsEVs isolated by the three methods(Method 1: Differential ultracentrifugation;Method 2: Method 1 +0.22 μm filtration;Method 3: Method 2 + two-step differential centrifugation step)under the condition of different enzyme concentrations(Collagenase D: 1,2,4 mg/ml;Deoxyribonuclease I(DNase I): 20,40,80 U/ml)and incubation time(20,30 min)were performed by Nano FCM.2.The expression of CD9 and CD63 in tdsEVs extracted by method 3 under the condition of different enzyme concentration and incubation time was detected by Nano FCM and Western blot.3.Cell uptake and migration assay of human umbilical vein endothelial cells(HUVECs)were performed with tdsEVs obtained by method 3 isolation protocol and the tissue treatment conditions of 20(1)(Collagenase D(4 mg/ml),DNase I(80 U/ml)and incubation time(20 min)).4.Three different sources of sEVs(cedsEVs,fresh and frozen tdsEVs)were analyzed by Nano FCM and Western blot for quantity(particle concentration and protein content),particle size,quality(purity and morphological appearance)and protein markers.5.Graph Pad Prism software was applied,and One-way ANOVA and unpaired two-tailed Student’s t test were used for statistical analysis.Results:1.TdsEVs extracted by method 3 had high purity and low coefficient of variation(CV)% values,while tdsEVs obtained by methods 1 and 2 had low purity as well as high CV% values for sEVs obtained by method 1.2.Method 3 obtained the highest level of protein marker for tdsEVs,CD9,under the condition of 20(1)combination whereas CD63 was not significantly different compared to other combinations.3.The tdsEVs obtained by method 3 under 20(1)combination conditions were incorporated into HUVECs,and the wound scratch assay showed that tdsEVs were capable of inhibiting the migration rate of HUVECs.4.Fresh tdsEVs are significantly better than cedsEVs in terms of particle number and protein content.Meanwhile,tdsEVs have a better size distribution and purity.However,fresh and frozen tdsEVs are not significantly different in terms of the foregoing four factors.5.Nano FCM and Western blot showed that the percentage of CD63-positive sEVs and CD63 content were significantly higher in cedsEVs than in fresh and frozen tdsEVs.6.Western blot showed that protein content of CD9,TSG101 and HSP70 in fresh and frozen tdsEVs were significantly higher than that in cedsEVs.7.Coomassie blue staining indicated that the total protein content of tdsEVs was higher than that of cedsEVs.Conclusion:We developed a convenient method for the enrichment of sEVs,and optimized the conditions for the isolation and purification of sEVs from liver cancer tissue.Our data suggest that an optimal combination(collagenase D of 4 mg/ml,DNase I of 80 U/ml and incubation time of 20 min)are key factors for isolating high quality sEVs from liver cancer tissues.