Extraction and identification of synovial tissue-derived exosomes by different separation techniques

Abstract

Objective: The aim of this study is to compare the efficiency of different separation techniques for extracting synovial tissue-derived exosomes. Methods: The synovial tissue discarded during knee arthroscopy or total knee arthroplasty surgery was collected from the Third Affiliated Hospital of Beijing University of Chinese Medicine. Ultracentrifugation (UC), filtration combined with size exclusion chromatography (SECF), and 8% polyethylene glycol (PEG) were used to extract synovial tissue-derived exosomes. Transmission electron microscopy (TEM), nanoparticle tracer analysis (NTA), and Western Blot (WB) were used to detect the morphology, particle size, and biomarker proteins (CD9, CD63, Flotillin-1, and calnexin) of exosomes. Results: The extracts of enriched round and discoid vesicles were successfully extracted with UC, SECF, and PEG. The results of TEM have shown that all three extraction methods can extract circular or elliptical vesicles with discand cup-shaped structures from the synovial tissue, with the diameter is about 30-150 nm. NTA suggested the main peaks of three groups of exosomes are around 100-120 nm, and the concentration of the three groups of exosomes was greater than 1 × 10 10 /ml. The results of WB showed that three positive protein markers (CD9, CD63, and Flotillin-1) were highly expressed in the suspension extracted by the three methods and low in the synovial tissue. However, the negative protein (calnexin) was highly expressed in synovial tissues and PEG group, while low in UC and SECF group. Conclusion: Morphology, particle size, and labeled protein marker detection confirmed that UC, SECF, and PEG can extract exosomes derived from synovial tissue; UC and SECF are more recommended for the extraction of synovial tissue-derived exosomes, which provides a methodological basis for studying the function and mechanism of synovial tissue exosomes in the future.

A Review of Exosomal Isolation Methods: Is Size Exclusion Chromatography the Best Option?

“…Since a single exosome isolation technique cannot reach optimal yield and purity, coupling SEC with other isolation methods including dUC, UF, or PEG-based retrieval can precipitate intact, highly purified exosomes in a reproducible manner. However, the limitations of these approaches as detailed above will then accompany the chimeric protocol [ 32 , 37 , 44 , 49 , 90 , 101 , 102 , 103 ].…”

Cinnamic Aldehyde Inhibits Lipopolysaccharide-Induced Chondrocyte Inflammation and Reduces Cartilage Degeneration by Blocking the Nuclear Factor-Kappa B Signaling Pathway

“…A loading buffer was then added, mixed well, exposed to a metal bath at 95°C for 5 min, and stored at −80°C after dispensing. Western blot (WB) analysis was performed in accordance with a method described previously ( Chen et al., 2019 ; Chen et al., 2020 ). Briefly, equal amounts of protein (40 μg) were separated using sodium-dodecyl polyacrylamide gel electrophoresis, loaded onto the 8%–12% separation gel according to the target molecular weight, and then transferred onto a polyvinylidene fluoride (PVDF) membrane.…”

Exosomes: roles and therapeutic potential in osteoarthritis

“…Chen et al compared the efficiency of three separation techniques (ultracentrifugation, filtration combined with size exclusion chromatography and 8% polyethylene glycol) for extracting synovial tissue-derived exosomes (Syn-Exos). 261 They found that the efficiency of exosome isolation differed among these three isolation methods and recommended ultracentrifugation and filtration combined with size exclusion chromatography for the extraction of Syn-Exos. 261 Therefore, additional measures are needed to optimize and standardize the existing separation methods so that the clinical use of MSC-Exos is safe and convenient.…”