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. 2020 Mar 9;15(1):97.
doi: 10.1186/s13018-020-01604-x.

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

Pu Chen  1 , Anmin Ruan  1 , Jun Zhou  1 , Liuwei Huang  2 , Xiaozhe Zhang  1 , Yufeng Ma  1 , Qingfu Wang  3
Affiliations

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

Pu Chen et al. J Orthop Surg Res. .

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 disc- and 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 × 1010/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.

Keywords: Exosomes; Extracellular vesicles; Osteoarthritis; Synovial tissues.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Morphological characteristics of synovial tissue-derived exosomes extracted by three different extraction methods. a The PEG group. b The UC group. c The SECF group
Fig. 2
Fig. 2
Morphological characteristics of synovial tissue-derived exosomes extracted by three different extraction methods. a The PEG group. b The UC group. c The SECF group
Fig. 3
Fig. 3
The protein concentration of synovial tissue-derived exosomes. *, compared with the UC group, P < 0.05
Fig. 4
Fig. 4
The exosomes biomarker detection results with WB. ST synovial tissues
See this image and copyright information in PMC

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