The comparative study on the extraction methods of exosomes from synovial fibroblasts in patients with rheumatoid arthritis / 中华风湿病学杂志

ABSTRACT

Objective:To compare the efficiency and purity of exosomes extracted from synovial fibroblasts of patients with rheumatoid arthritis by ultracentrifugation, size exclusion chromatography and modified polymer precipitation.Methods:The exosomes were extracted from human synovial fibroblasts by ultracentrifugation, size exclusion chromatography and modified polymer precipitation. Transmission electron microscopy, particle size detection and western Blot were used to identify the morphological characteristics, particle size distribution, concentration, and expression of marker proteins. One-way analysis of variance (ANOVA) was used for comparison among the three groups, and LSD- t test was used for pairwise comparison. P<0.05 was considered statistically significant. Results:Exosomes could be successfully obtained with all three extraction methods. The typical "saucer-like" structure could be observed under transmission electron microscope. The marker proteins of exosomes TSG101, Syntenin-1 and CD63 were all detectable by western blot. The peaks of main particle size were located within 30~150 nm. As for purity, the exosomes obtained by ultracentrifugation showed the highest purity, while modified polymer precipitation was the worst, with a large number of polymer particles and impurities protein. The purity of exosomes obtained by size exclusion chromatography was the moderate. For extraction efficiency, concentrations of exosomes particles obtained by the three methods were different ( F=9.61, P=0.049), and modified polymer precipitation was significantly higher than ultracentrifugation in terms of concentration of exosomes particles [(98.0±17.0)×10 10 particles/ml vs (11.6±7.7)×10 10 particles/ml, t=-4.34, P=0.023]. Conclusion:Human synovial fibroblasts derived exosomes canbe obtained by three methods. Ultracentrifugation is time-consuming, but can produce high-purity exosomes, which may be considered in the situation when high purity requirement with large volume samples are needed. Size exclusion chromatography is a good choice with high yield and purity exosomes, and suitable for small volume samples. Modified polymer precipitation is not recommended due to production of lowest purity exosomes.