Extraction and quality evaluation of exosomes from joint effusion

Objective: The aim of this study was to compare the quality and efficiency of exosomes extracted from knee joint effusion by different methods, laying a foundation for further research on exosomes from knee joint effusion. Methods: : To separate and extract joint exosomes by 8% polyethylene glycol (PEG) precipitation method, ultracentrifugation method (UC) and ultrafiltration with exclusion chromatography (SECF). Transmission electron microscopy (TEM) and nanoparticle tracing technology (NTA) were used to detect particle morphology and particle size, and Western Blot (WB) was used to detect marker proteins of granule surface (CD9, CD63, Flotillin-1 and calnexin). Results: : Three methods separated vesicle-like round particles from joint effusion successfully. The results of TEM show that the particles obtained by the three extraction methods are round or oval vesicles, the plasma membrane is intact, the size is different, and the diameter is distributed between 30 and 150 nm. Compared with SECF group, PEG group had more background particle impurities. and the broken vesicle fragments can be seen in the UC group; The results of NTA show that the main peaks of the three groups of particles are between 100-120nm, and the particle concentration is greater than 1×10 10 /ml; The results of WB show that the protein expressions of CD9, CD63 and Flotillin-1 in the suspension extracted by the three methods were higher, the expression of calnexin protein in the PEG group was higher than the UC group and SECF group. Conclusion: The three extraction methods can extract the exosomes of joint effusion successfully. The quality of exosomes obtained by the SECF method is relatively high, while the PEG precipitation method contains a small amount of impurity particles. UC method does not guarantee the integrity of exosomes. In summary, we recommend the SECF method to extract and isolate joint effusion exudates-derived exosomes when further studying the role of exosomes in the pathogenesis of knee osteoarthritis.