| Exosomes are a kind of micro vesicles secreted by biological cells.Because of its unique biological characteristics and extensive functions,exosomes are gradually used in the field of cosmetics and new drugs.However,the separation,purification and characterization methods are not perfect and need to be further optimized and studied.Using exosomes that originated from human urine and human renal epithelial 293 cells.Different extraction and purification methods were established for exosomes from different sources.The urine was roughly separated by different centrifugation,and then the exosomes were extracted by 700 kD molecular sieve chromatography.The mobile phase was phosphate buffer(20 mmol/L)with pH=7.0.The purity of exosomes after separation and purification was more than 98%.In addition to the separation and purification method of urinary exosomes,human renal epithelial 293 cell-derived exosomes also can be separated by ion exchange chromatography and then molecular sieve chromatography.The final purity also can reach 98%.The purity of exosomes can be characterized by molecular sieve liquid chromatography.The phosphate system(20 mmol/L)is used as the mobile phase and analyzed in Agilent 1000(?)column at the flow rate of 0.3ml/min.The results show that there is only one chromatographic peak in the peak diagram of pure exosomes,and its retention time is about 9.1 min.The sample conforms to the typical morphology of exosomes by transmission electron microscope.That the single peak is the peak of exosomes.When the protein concentration of the exosomes is about 100μg/ml,the high of the purity peak is about 10 mAU.And the purity peak shape of the exosomes is better.The BCA method was verified.The linear R~2is 0.9981 and the accuracy recovery is greater than90%,so the method met the requirements of determination.The morphological observation conditions of exosomes were further optimized.When the negative staining time of staining solution(2%uranyl acetate)was 30 s,the staining effect of exosomes was the best.The morphology was clear and the background was bright.The particle size distribution of exosomes was analyzed by TEM,nano flow cytometry and nano particle tracking analysis(NTA).The median particle size distribution of TEM and nano flow cytometry was about70 nm,which was far from the 138.5 nm particle size of NTA.The charged characteristics of exosomes were further studied.Four different exosome groups were separated by ion exchange chromatography.Their purity,zeta potential,morphology and particle size distribution were characterized respectively.The purity was83.68%,84.90%,48.02%and 32.19%.The zeta potential was-12.13 mV,-12.97 mV,-14.36 mV and-12.84 mV.The particle size distribution was 69.25 nm,71.75 nm,72.75nm and 72.75 nm.Different exosomes have different charge characteristics,continuous distribution and different charged strength. |
