| Tissue types | Collection and pre-processing | EV characterization | Method of analysis | Key findings | Citation | Author | 影响因子 | 备注 |
| 组织处理 | Pre-EV isolation | EV-isolation | Methods | Markers | PMID | Year |
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| 新鲜人/动物组织 |
| 2500g, 30min;
10000g, 35min; | 0.2 μm过滤;
110000g, 2h; | NTA, WB, TEM, IEM, fluorescence staining, FCM | CD9, CD63, CD8,
ESCRT proteins (Alix, TSG101) | / | EVs分离鉴定方法; | 27801511 | 2016 |
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| 人转移性黑色素瘤组织 | 新鲜组织切碎到1-2mm;
在含有collagenase D (2 mg/ml)和DNase I (40 U/ml)的RPMI1640培养基中37°C孵育30min; | 70 μm过滤;
300g, 10min;
2000g, 20min;
16500g, 20min; | 118000g, 2.5h; | TEM, NTA, WB | CD9, CD63, CD81, calnexin, flotillin-1 | NanoLC-MS/MS analysis | 分离EVs亚型方法;
ADAM10富集于LD EVs;
mitofilin富集于大EVs; | 32128073 | 2020 | Crescitelli et al. |
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| 人黑色素瘤组织 | 新鲜组织切碎到2mm;
在含有collagenase D (2 mg/ml)和DNase I (40 U/ml)的RPMI1640培养基中37°C孵育30min，期间24rpm缓慢振荡; | 300g, 10min;
2000g, 20min;
16500g, 20min; | 118000g, 2.5h;
碘克沙醇密度梯度离心, 186000g, 16h; | TEM, WB, ExoView analysis | CD63, CD81, CD9, flotillin-1, Calnexin | RNA profile analysis, ExoView, proteomic analysis | 人黑色素瘤组织外泌体分离鉴定方案; | 33495626 | 2021 | Crescitelli et al. |
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| 新鲜人肠组织 | 组织切碎到<0.5cm;
含collagenase I(300U/ml)的HBSS中孵育30min，期间缓慢振荡;
用含有蛋白酶抑制剂的PBS终止消化； | 70 μm过滤;
1000g, 10min;
2000g, 20min;
5000g, 30min;
15000g, 1h;
0.2 μm PVDF过滤; | 120000g, 130min;
碘克沙醇密度梯度离心, 288000g, 5h;
100000g, 70min; | NTA, TEM, WB | CD9, CD63, CD81, Alix, Tsg101 | RT-qPCR | 分离肠组织外泌体并应用于肠缺血-再灌注(I/R)损伤研究; | 32090587 | 2020 |
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| 人肾癌及癌旁组织 | 组织切碎;
加入4ml DMEM，4°C孵育1h； | 2000g, 20min;
16000g, 20min; | 100000g, 90min;
100000g, 90min; | WB, TEM, NTA | CD9, CD63, CD81 | Quantitative LC/MS analysis | Te‐EVs;
蛋白组分析;
AZU1 | 28975613 | 2018 |
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| 人转移性黑色素瘤组织 | 组织切碎到1-2mm;
在含有collagenase D (2 mg/ml)和DNase I (40 U/ml)的RPMI1640培养基中37°C孵育30min; | 70 μm过滤;
300g, 10min;
2000g, 20min;
16500g, 20min; | 110000g, 2.5h; | TEM, WB, ELISA, particle measurement | CD9, CD81 | MS analysis, RNA detection | 黑色素瘤组织外泌体富含线粒体膜蛋白; | 31497264 | 2019 |
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| 尸检脑组织 | 组织冰上切碎，冰上解冻；
在含有collagenase III (75 U/mL)的Hibernate-E培养基中37°C孵育20min，期间缓慢振荡;
加入蛋白酶抑制剂(PI/PS)终止消化; | 37°C水浴振荡20min;
300g, 5min;
2000g, 10min;
10000g, 5min; | Triple sucrose cushion;
180000g, 3h; | TEM, WB, IB | Calnexin | Small RNA sequencing | AD脑组织EVs miRNA分析; | 32922692 | 2020 |
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| 毫米大小癌及癌旁组织 | 组织切碎;
在含有双抗的RPMI1640培养基中37°C孵育24h; | 500g, 10min;
3000g, 20min;
12000g, 20min; | 100000g, 70min;
1 ml sucrose density cushion,
100000g, 70min; | WB, TEM, NTA | CD9, CD81, TSG101 | MS蛋白组 | pan-EVP markers (ACTB, MSN, RAP1B);
肿瘤特异性EVP蛋白; | 32795414 | 2020 |
| 66 |
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| 小鼠黑色素瘤组织 | 组织切碎到<3mm, PBS洗一遍;
500g, 4min;
组织解离为单细胞悬液(含有25 μg/ml DNase I, 5 Wünsch units of Liberase的PBS，37°C，20min，缓慢振荡)；
70μm过滤;
PBS(含5 mM EDTA，25 μg/ml DNase I)洗一遍； |
500g, 10min;
500g, 10min;
2000g, 15min;
2000g, 15min; | 16.5K EVs:
16500g, 24min (×2);
100K EVs: 100000g,70min (×2); | TEM, WB | Calnexin, cytochrome-C, GM130 | LC-MS/MS, transcriptomics analysis | Cells from two types of melanoma phenocopy migratory behaviour through EV exchange. | 29907695 | 2018 |
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| 结直肠癌及癌旁组织 | 新鲜组织冰上切碎;
在含有collagenase I (250 units/ml) RPMI1640 培养基中37°C孵育30min;
置于冰上，加入含多种蛋白酶抑制剂的PBS；
吹散细胞； | 60mm筛网;
40mm筛网;
400g, 10min;
2000g, 20min;
15000g, 40min;
0.22 μm PES过滤; | 120000g, 4h (×2);
碘克沙醇密度梯度离心; | IB | CD63, CD81, Syntenin-1, Calreticulin | MS, RNA sequencing, DNA analysis, direct immunoaffinity Capture | CD9, CD63, CD81, Annexin V在外泌体中缺失;
Annexin A1和A2为非外泌体EVs新markers; | 30951670 | 2019 |
| 66 |
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| 小鼠结直肠癌组织 | PBS洗一遍;
组织在含2 mM EDTA的PBS中切碎; | 70μm细胞筛网;
500g, 5min;
3000g, 10min;
10000g, 30min; | 110000g, 70min;
134000g, 70min; | TEM, NTA, WB, Flow cytometry | CD9, CD81, ALIX | LC-MS/MS, miRNA profiling, Lipidome analysis | TAM-EVs诱导炎症及抗肿瘤反应; | 31167148 | 2019 |
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| AD小鼠脑组织 | 在Hibernate-E培养基中37°C孵育20min;
研磨(loose-fit Dounce homogenizer); | 500g, 5min;
2000g, 10min;
10000g, 30min;
0.45 μm 过滤; | 100000g, 2h;
1.5 ml of 0.25 M sucrose buffer for gradient purification; floatation density gradient. | WB | CD63, CD81, Alix, TSG101, HSC70, Rab8a | MS, enrichment analysis | Ti-EVs分离方法; | 29894726 | 2018 |
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| 人脑组织 | 在Hibernate-E培养基(木瓜蛋白酶,20 units/ml)中37°C孵育15min; | 40 μm 筛网;
300g, 10min;
2000g, 10min;
10000g, 10min;
0.22 μm 过滤; | 100000g, 70min;
0.475 M of sucrose solution | NTA, TEM | / | Label-free Nano-LC-MS/MS analysis | Tau、Aβ1-42上调; | 32301581 | 2020 |
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| 人脑组织 | PBS中切碎，涡旋; | 300g, 10min;
1200g, 10min(×2);
0.22 μm 过滤;
10000g, 30min(×2); | 22000g, 22h; | WB, TEM, Immunogold Labeling, | CD63, GAPDH, flotillin-2 | MiRNA expression analysis, qPCR analysis | SZ样品miR-497上调;
BD样品miR-29c上调; | 23382797 | 2013 |
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| 恒河猴脑组织 | 在Hibernate-A培养基(木瓜蛋白酶,20 units/ml)中37°C振荡孵育15min; | 40 μm 筛网;
5 μm 筛网;
0.22 μm 筛网;
300g, 10min;
2000g, 10min;
10000g, 30min; | 100000g,60min (×2);
2 ml of 0.95 M sucrose solution and inserted inside a sucrose step gradient column; 200000g,16h; | TEM, WB | CD9, CD63, CD81, HSP70, flotillin, TSG101 | Small RNA sequencing, qRT-PCR | Neurotoxicity triggered by EV-miR-21 was not influenced by apoptosis inhibition but restricted by necrostatin-1. | 26154133 | 2015 |
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| 小鼠脑组织 |
| 40 μm 筛网;
0.2 μm 筛网;
300g, 10min;
2000g, 10min;
10000g, 30min; | 100000g,70min (×2);
2 ml of 0.95 M sucrose solution centrifuged at 200000g for 16 h. | Immunoelectron microscopy | TSG101 | Gene expression analysis, image analysis | 小神经胶质细胞通过外泌体扩散tau; | 26436904 | 2015 |
| 28 |
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| 人脑组织 | 冰冻组织冰上切碎;
在Hibernate-E培养基(collagenase III ，75 U/ml )中37°C振荡孵育20min;
蛋白酶抑制剂终止消化(PhosSTOP和Complete Protease Inhibitor); | 300g, 15min;
2000g, 15min;
0.2 μm 筛网;
10000g, 30min; | Sucrose density gradient ultracentrifugation (SDGU);
110000g,70min;
ultrafiltration through a 10 kDa MWCO protein concentrator. | TEM, NTA, WB, NanoFCM flow analysis | CD9, CD63, CD81, Rab27, TSG101, Syntenin, Calnexin, GM130 | Small RNA sequencing, MS | 3个物种脑组织Ti-EVs分离比较，强调了不同物种的分离参数; | 32944174 | 2020 |
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| 人脂肪组织 |
| 800g, 10min;
2000g, 10min;
12000g, 30min;
0.2 μm 筛网; | 100000g, 2h; | Fluorescence NTA, WB, EM | CD9, CD63, TSG101, Grp94 (a negative control) | MS analysis, Ingenuity pathway analysis | Adipose Ti-EVs mediate changes in placental functions in GDM and are involved in some pregnancy complications. | 30517676 | 2019 |
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| 脂肪组织 | 切碎至1-2mm;
加入到含双抗的α-MEM培养基中，37°C 100rpm/min振荡孵育2天； | 2000g, 10min;
5000g, 30min; | 5000g, 30min;
Exosome Isolation TM reagent 4°C孵育过夜;
10000g, 1h; | WB, TEM, Zetasizer Nano ZS | CD9, CD63, ALIX, TSG101 | MS analysis, Bioinformatic analysis, Real-time PCR | NPM3, STEAP3, DAD1与脂肪形成 | 32597661 | 2020 |
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| Human obese white adipose tissue explant |
| 1800g, 5min;
0.22 μm 筛网;
10000g, 20min; | 100000g,90min (×2); | TEM, NTA, IB | CD9, CD63, CD81, negative control GRP94 | MS DDA qualitative analysis | Obese AT release functional EVs carrying AT and obesity-specific biomarkers depending on the original AT. | 33465489 | 2022 |
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| 小鼠脂肪组织 | 切碎至>4mm;
加入到含抗生素和10%FBS(已去除外泌体)的DMEM培养基中，37°C 培养； | 200g, 10min;
500g, 10min(×2);
2000g, 15min;
10000g, 30min; | 70000g, 60min;
5 ml of 2.6 M sucrose at 270000g 16 h;
Gradient fractions were centrifugated at 70000g 1h. | / | / | Fluorescence activated cell sorter (FACS) analysis | Adipose Ti-ELVs mediate crosstalk between AT and macrophages; ObELV induced TNF-α and IL-6 activation and insulin resistance require the TLR4/TRIF pathway. | 19675137 | 2009 |
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| 小鼠脂肪组织 | 灌洗以去除组织中血液;
切碎;
37°C解离组织1h(100 mM HEPES, 1.5% BSA, 5 mM glucose, 1 mM calcium and 1mg/ml collagenase D, 2.4U/ml dispase II);
2 mM EGTA中止消化; | 100 μm 筛网;
600g, 5min;
1200g, 15min;
10000g, 15min;
0.22 μm 筛网; | 100000g, 90min (×2); | NTA, TEM, WB | CD63, Alix, TSG101 | Proteomics analysis, LC‐MS lipidomics analysis. | Adipose Ti‐EVs are involved in the response to changes in systemic nutrient conditions. | 30293865 | 2018 | 66 |
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| 小鼠脂肪组织 | 剪碎；
1000g，5min，清洗组织；
培养基中37 °C孵育30min；
换液，培养基中37 °C孵育2h以释放外泌体； | 1000g for 5 min at room temperature and re‐dissolved in medium; incubated for 30 min at 37°C, 5% CO2. | Medium exchange, tissues were incubated for 2 h at 37°C, 5% CO2. The supernatant was used for EVs isolation according to the manufacturer's instruction. | WB, TEM | CD63, Hsp70, CytoC, α‐Tubulin | MiRNA profiling | 外泌体miR‐92a与人BAT活性负相关; | 27117818 | 2016 |
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| 小鼠肝组织 | 通过灌注胶原酶来解离肝脏组织； | 肝灌注;
70 μm 筛网;
50g, 10min;
300g, 10min;
2000g, 20min;
10000g, 70min; | 100000g, 70min (×2); | NTA, tunable resistive pulse sensing | / | / | An optimum and replicable procedure for the isolation of hepatic Ti‐EVs. | 31498323 | 2019 |
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| JOVE |