| Tissue types | Collection and pre-processing | EV characterization | Method of analysis | Key findings | Citation | Author | 影響因子 | 備注 |
| 組織處理 | Pre-EV isolation | EV-isolation | Methods | Markers | PMID | Year |
|
|
|
| 新鮮人/動物組織 |
| 2500g, 30min;
10000g, 35min; | 0.2 μm過濾;
110000g, 2h; | NTA, WB, TEM, IEM, fluorescence staining, FCM | CD9, CD63, CD8,
ESCRT proteins (Alix, TSG101) | / | EVs分離鑒定方法; | 27801511 | 2016 |
|
|
|
| 人轉移性黑色素瘤組織 | 新鮮組織切碎到1-2mm;
在含有collagenase D (2 mg/ml)和DNase I (40 U/ml)的RPMI1640培養基中37°C孵育30min; | 70 μm過濾;
300g, 10min;
2000g, 20min;
16500g, 20min; | 118000g, 2.5h; | TEM, NTA, WB | CD9, CD63, CD81, calnexin, flotillin-1 | NanoLC-MS/MS analysis | 分離EVs亞型方法;
ADAM10富集于LD EVs;
mitofilin富集于大EVs; | 32128073 | 2020 | Crescitelli et al. |
|
|
| 人黑色素瘤組織 | 新鮮組織切碎到2mm;
在含有collagenase D (2 mg/ml)和DNase I (40 U/ml)的RPMI1640培養基中37°C孵育30min，期間24rpm緩慢振蕩; | 300g, 10min;
2000g, 20min;
16500g, 20min; | 118000g, 2.5h;
碘克沙醇密度梯度離心, 186000g, 16h; | TEM, WB, ExoView analysis | CD63, CD81, CD9, flotillin-1, Calnexin | RNA profile analysis, ExoView, proteomic analysis | 人黑色素瘤組織外泌體分離鑒定方案; | 33495626 | 2021 | Crescitelli et al. |
|
|
| 新鮮人腸組織 | 組織切碎到<0.5cm;
含collagenase I(300U/ml)的HBSS中孵育30min，期間緩慢振蕩;
用含有蛋白酶抑制劑的PBS終止消化； | 70 μm過濾;
1000g, 10min;
2000g, 20min;
5000g, 30min;
15000g, 1h;
0.2 μm PVDF過濾; | 120000g, 130min;
碘克沙醇密度梯度離心, 288000g, 5h;
100000g, 70min; | NTA, TEM, WB | CD9, CD63, CD81, Alix, Tsg101 | RT-qPCR | 分離腸組織外泌體并應用于腸缺血-再灌注(I/R)損傷研究; | 32090587 | 2020 |
|
|
|
| 人腎癌及癌旁組織 | 組織切碎;
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;
蛋白組分析;
AZU1 | 28975613 | 2018 |
|
|
|
| 人轉移性黑色素瘤組織 | 組織切碎到1-2mm;
在含有collagenase D (2 mg/ml)和DNase I (40 U/ml)的RPMI1640培養基中37°C孵育30min; | 70 μm過濾;
300g, 10min;
2000g, 20min;
16500g, 20min; | 110000g, 2.5h; | TEM, WB, ELISA, particle measurement | CD9, CD81 | MS analysis, RNA detection | 黑色素瘤組織外泌體富含線粒體膜蛋白; | 31497264 | 2019 |
|
|
|
| 尸檢腦組織 | 組織冰上切碎，冰上解凍；
在含有collagenase III (75 U/mL)的Hibernate-E培養基中37°C孵育20min，期間緩慢振蕩;
加入蛋白酶抑制劑(PI/PS)終止消化; | 37°C水浴振蕩20min;
300g, 5min;
2000g, 10min;
10000g, 5min; | Triple sucrose cushion;
180000g, 3h; | TEM, WB, IB | Calnexin | Small RNA sequencing | AD腦組織EVs miRNA分析; | 32922692 | 2020 |
|
|
|
| 毫米大小癌及癌旁組織 | 組織切碎;
在含有雙抗的RPMI1640培養基中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蛋白組 | pan-EVP markers (ACTB, MSN, RAP1B);
腫瘤特異性EVP蛋白; | 32795414 | 2020 |
| 66 |
|
| 小鼠黑色素瘤組織 | 組織切碎到<3mm, PBS洗一遍;
500g, 4min;
組織解離為單細胞懸液(含有25 μg/ml DNase I, 5 Wünsch units of Liberase的PBS，37°C，20min，緩慢振蕩)；
70μm過濾;
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 |
|
|
|
| 結直腸癌及癌旁組織 | 新鮮組織冰上切碎;
在含有collagenase I (250 units/ml) RPMI1640 培養基中37°C孵育30min;
置于冰上，加入含多種蛋白酶抑制劑的PBS；
吹散細胞； | 60mm篩網;
40mm篩網;
400g, 10min;
2000g, 20min;
15000g, 40min;
0.22 μm PES過濾; | 120000g, 4h (×2);
碘克沙醇密度梯度離心; | IB | CD63, CD81, Syntenin-1, Calreticulin | MS, RNA sequencing, DNA analysis, direct immunoaffinity Capture | CD9, CD63, CD81, Annexin V在外泌體中缺失;
Annexin A1和A2為非外泌體EVs新markers; | 30951670 | 2019 |
| 66 |
|
| 小鼠結直腸癌組織 | PBS洗一遍;
組織在含2 mM EDTA的PBS中切碎; | 70μm細胞篩網;
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誘導炎癥及抗腫瘤反應; | 31167148 | 2019 |
|
|
|
| AD小鼠腦組織 | 在Hibernate-E培養基中37°C孵育20min;
研磨(loose-fit Dounce homogenizer); | 500g, 5min;
2000g, 10min;
10000g, 30min;
0.45 μm 過濾; | 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分離方法; | 29894726 | 2018 |
|
|
|
| 人腦組織 | 在Hibernate-E培養基(木瓜蛋白酶,20 units/ml)中37°C孵育15min; | 40 μm 篩網;
300g, 10min;
2000g, 10min;
10000g, 10min;
0.22 μm 過濾; | 100000g, 70min;
0.475 M of sucrose solution | NTA, TEM | / | Label-free Nano-LC-MS/MS analysis | Tau、Aβ1-42上調; | 32301581 | 2020 |
|
|
|
| 人腦組織 | PBS中切碎，渦旋; | 300g, 10min;
1200g, 10min(×2);
0.22 μm 過濾;
10000g, 30min(×2); | 22000g, 22h; | WB, TEM, Immunogold Labeling, | CD63, GAPDH, flotillin-2 | MiRNA expression analysis, qPCR analysis | SZ樣品miR-497上調;
BD樣品miR-29c上調; | 23382797 | 2013 |
|
|
|
| 恒河猴腦組織 | 在Hibernate-A培養基(木瓜蛋白酶,20 units/ml)中37°C振蕩孵育15min; | 40 μm 篩網;
5 μm 篩網;
0.22 μm 篩網;
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 |
|
|
|
| 小鼠腦組織 |
| 40 μm 篩網;
0.2 μm 篩網;
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 | 小神經膠質細胞通過外泌體擴散tau; | 26436904 | 2015 |
| 28 |
|
| 人腦組織 | 冰凍組織冰上切碎;
在Hibernate-E培養基(collagenase III ，75 U/ml )中37°C振蕩孵育20min;
蛋白酶抑制劑終止消化(PhosSTOP和Complete Protease Inhibitor); | 300g, 15min;
2000g, 15min;
0.2 μm 篩網;
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個物種腦組織Ti-EVs分離比較，強調了不同物種的分離參數; | 32944174 | 2020 |
|
|
|
| 人脂肪組織 |
| 800g, 10min;
2000g, 10min;
12000g, 30min;
0.2 μm 篩網; | 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 |
|
|
|
| 脂肪組織 | 切碎至1-2mm;
加入到含雙抗的α-MEM培養基中，37°C 100rpm/min振蕩孵育2天； | 2000g, 10min;
5000g, 30min; | 5000g, 30min;
Exosome Isolation TM reagent 4°C孵育過夜;
10000g, 1h; | WB, TEM, Zetasizer Nano ZS | CD9, CD63, ALIX, TSG101 | MS analysis, Bioinformatic analysis, Real-time PCR | NPM3, STEAP3, DAD1與脂肪形成 | 32597661 | 2020 |
|
|
|
| Human obese white adipose tissue explant |
| 1800g, 5min;
0.22 μm 篩網;
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 |
|
|
|
| 小鼠脂肪組織 | 切碎至>4mm;
加入到含抗生素和10%FBS(已去除外泌體)的DMEM培養基中，37°C 培養； | 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 |
|
|
|
| 小鼠脂肪組織 | 灌洗以去除組織中血液;
切碎;
37°C解離組織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 篩網;
600g, 5min;
1200g, 15min;
10000g, 15min;
0.22 μm 篩網; | 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 |
|
|
| 小鼠脂肪組織 | 剪碎；
1000g，5min，清洗組織；
培養基中37?°C孵育30min；
換液，培養基中37?°C孵育2h以釋放外泌體； | 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 | 外泌體miR‐92a與人BAT活性負相關; | 27117818 | 2016 |
|
|
|
| 小鼠肝組織 | 通過灌注膠原酶來解離肝臟組織； | 肝灌注;
70 μm 篩網;
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 |
|
| JOVE |