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

技术手段	原理	优点	缺点	时间/h	纯度	体积/mL	产量
超速离心	尺寸和密度不均匀的成分具有不同的沉积速度	应用广,适合大批量样品,成本低,技术成熟	耗时长,操作烦琐,可能损伤外泌体	>4	中等	≤25	低
密度梯度离心	尺寸和密度不均匀的成分具有不同的沉积速度	纯度高,避免外泌体损伤	耗时长,操作烦琐,产率低	>16	高	≤25	低
超滤	利用膜微孔筛选尺寸不同的分子	简单,无须专用设备和试剂	过滤膜堵塞,颗粒直径较小的外泌体流失	<4	高	≤15	高
尺寸排阻色谱法	利用不同尺寸颗粒分子通过膜的时间差异	简单、经济、保持生物功能和结构	需要特殊的色谱柱和填料,脂蛋白污染	0.3左右	高	0.4~1.5	高
免疫亲和层析	利用特定抗体或配体进行高度特异性分离	高特异性外泌体亚型分离	价格昂贵,取决于抗体的特异性	4~20	高	0.4~3	中等

Table 1 Comparison of common exosomes isolation methods and their advantages /disadvantages^[18⇓-20]

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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
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 2 Summary of exosome isolation on different body fluid types^[57]
Table 3 Exosomal non-coding RNAs as diagnostic/prognostic markers^[74⇓-76]

模态框（Modal）标题

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