Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, Australia.
Proteomics. 2013 Nov;13(22):3354-64. doi: 10.1002/pmic.201300282. Epub 2013 Oct 18.
Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including blood. Recent studies have highlighted the critical application of exosomes as personalized targeted drug delivery vehicles and as reservoirs of disease biomarkers. While these research applications have created significant interest and can be translated into practice, the stability of exosomes needs to be assessed and exosome isolation protocols from blood plasma need to be optimized. To optimize methods to isolate exosomes from blood plasma, we performed a comparative evaluation of three exosome isolation techniques (differential centrifugation coupled with ultracentrifugation, epithelial cell adhesion molecule immunoaffinity pull-down, and OptiPrep(TM) density gradient separation) using normal human plasma. Based on MS, Western blotting and microscopy results, we found that the OptiPrep(TM) density gradient method was superior in isolating pure exosomal populations, devoid of highly abundant plasma proteins. In addition, we assessed the stability of exosomes in plasma over 90 days under various storage conditions. Western blotting analysis using the exosomal marker, TSG101, revealed that exosomes are stable for 90 days. Interestingly, in the context of cellular uptake, the isolated exosomes were able to fuse with target cells revealing that they were indeed biologically active.
外泌体是各种细胞释放的纳米囊泡，可在包括血液在内的体液中检测到。最近的研究强调了外泌体作为个性化靶向药物传递载体和疾病生物标志物储存库的关键应用。虽然这些研究应用引起了极大的兴趣，并可以转化为实践，但外泌体的稳定性需要进行评估，并且需要优化从血浆中分离外泌体的方案。为了优化从血浆中分离外泌体的方法，我们使用正常人血浆对三种外泌体分离技术（差速离心结合超速离心、上皮细胞黏附分子免疫亲和下拉法和 OptiPrep（TM）密度梯度分离）进行了比较评估。基于 MS、Western blot 和显微镜结果，我们发现 OptiPrep（TM）密度梯度法在分离纯净的外泌体群体方面更具优势，这些群体不含高丰度的血浆蛋白。此外，我们还评估了在不同储存条件下，外泌体在血浆中 90 天内的稳定性。使用外泌体标志物 TSG101 的 Western blot 分析显示，外泌体在 90 天内稳定。有趣的是，在细胞摄取的情况下，分离的外泌体能够与靶细胞融合，这表明它们确实具有生物活性。
