China Biotechnology

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Mammalian Exosomes: Isolation and Purification, Clinical Applications and Prospects

JIANG Shanshan, YIN Yuqi, LI Qianyu, ZHANG Liangyu, YANG Mei, WANG Yonghua

China Biotechnology, 2024, 44(12): 51-63. DOI: 10.13523/j.cb.2404028

样本类型	疾病类型	分离方法	外泌体验证
血浆	细胞瘤	超速离心法	尺寸:100 nm(透射电子显微镜) 蛋白质检测:CD63(免疫印迹法)
血浆	乳腺癌	超速离心法	蛋白质检测:CD9(免疫印迹法)
血浆	白血病	超速离心法,尺寸排阻色谱	尺寸:100 nm(透射电子显微镜) 蛋白质检测:CD81(免疫印迹法)
血清	乳腺癌	超速离心法,密度梯度离心	尺寸:100 nm(透射电子显微镜)
血清	胰腺癌	0.2 μm过滤,超速离心法,微流控技术(ExoChip)	尺寸:100 nm(透射电子显微镜) 蛋白质检测:CD63和TSG101(免疫印迹法)
腹水	乳腺癌、卵巢癌、肺癌	超速离心法,密度梯度离心(蔗糖)	尺寸:100 nm(透射电子显微镜) 蛋白质检测:CD9(免疫印迹法)
恶性胸腔积液	多种癌症(乳腺癌、肺癌、卵巢癌)	超速离心法,密度梯度离心	尺寸:100 nm(透射电子显微镜)
尿液	前列腺癌	超速离心法	蛋白质检测:Alix(免疫印迹法)

Table 2 Summary of exosome isolation on different body fluid types^[57]

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Fig.1 Schematic diagram of exosome biogenesis (by Figdraw) Exosomes are small membrane vesicles. They are formed as intraluminal vesicles by inward budding of the cell membrane into early endosomes, subsequently undergo migration and sorting to produce multivesicular body (MVB) and form intraluminal vesicles (ILVs). Upon fusion of thus arising MVB with the plasma membrane, these vesicles are released as exosomes
Table 1 Comparison of common exosomes isolation methods and their advantages /disadvantages^[18⇓-20]
Fig.2 Schematic diagram of the routine technique of exosome isolation (modified from the references[18], by Figdraw) A: Ultracentrifugation. Exosomes were obtained by multiple centrifugation, centrifugal forces from 300 g up to 100 000 g, after each centrifugation step, the supernatant including cells and cellular debris was removed and collected for subsequent centrifugation, the centrifugation is performed at 4℃ B: Density gradient ultracentrifugation. Preparation of an isometric density gradient ultracentrifugation by gradually decreasing the density layer from bottom to top. After prolonged centrifugation, exosomes and other extracellular components reach a static position in a medium of similar density to each component C: Size-exclusion chromatography. When passing a solution through a stationary phase consisting of porous resin particles, molecules can be separated according to size D: Immunoaffinity. After incubating exosome-containing liquids with exosome-specific antibody coupled to the solid substrates, exosomes can be enriched onto solid substrates
Table 3 Exosomal non-coding RNAs as diagnostic/prognostic markers^[74⇓-76]

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