Extracellular vesicles (EVs) are lipid bilayered membrane structures released by all cells. Most EV studies have been performed by using cell lines or body fluids, but the number of studies on tissue-derived EVs is still limited. Here, we present a protocol to isolate up to six different EV subpopulations directly from tissues. The approach includes enzymatic treatment of dissociated tissues followed by differential ultracentrifugation and density separation. The isolated EV subpopulations are characterized by electron microscopy and RNA profiling. In addition, their protein cargo can be determined with mass spectrometry, western blot and ExoView. Tissue-EV isolation can be performed in 22 h, but a simplified version can be completed in 8 h. Most experiments with the protocol have used human melanoma metastases, but the protocol can be applied to other cancer and non-cancer tissues. The procedure can be adopted by researchers experienced with cell culture and EV isolation.

细胞外囊泡 (EV) 是由所有细胞释放的脂质双层膜结构。大多数 EV 研究都是通过使用细胞系或体液进行的，但对组织衍生 EV 的研究数量仍然有限。在这里，我们提出了一个协议，可直接从组织中分离多达六个不同的 EV 亚群。该方法包括对解离组织进行酶处理，然后进行微分超速离心和密度分离。分离的 EV 亚群的特征在于电子显微镜和 RNA 分析。此外，它们的蛋白质货物可以用质谱法、蛋白质印迹法和 ExoView 来确定。组织-EV 隔离可在 22 小时内执行，但简化版本可在 8 小时内完成。该协议的大多数实验都使用了人类黑色素瘤转移瘤，但该协议可应用于其他癌症和非癌症组织。具有细胞培养和 EV 分离经验的研究人员可以采用该程序。