Wu Yueting, Deng Wentao, Klinke David J
Department of Chemical Engineering and Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, WV 26505, USA.
Analyst. 2015 Oct 7;140(19):6631-42. doi: 10.1039/c5an00688k.
As a type of secreted membrane vesicle, exosomes are an emerging mode of cell-to-cell communication. Yet as exosome samples are commonly contaminated with other extracellular vesicles, the biological roles of exosomes in regulating immunity and promoting oncogenesis remain controversial. Wondering whether existing methods could distort our view of exosome biology, we compared two direct methods for imaging extracellular vesicles and quantified the impact of different production and storage conditions on the quality of exosome samples. Scanning electron microscopy (SEM) was compared to transmission electron microscopy (TEM) as alternatives to examine the morphology of exosomes. Using SEM, we were able to distinguish exosomes from other contaminating extracellular vesicles based on the size distribution. More importantly, freezing of samples prior to SEM imaging made it more difficult to distinguish exosomes from extracellular vesicles secreted during cell death. In addition to morphology, the quality of RNA contained within the exosomes was characterized under different storage conditions, where freezing of samples also degraded RNA. Finally, we developed a new flow cytometry approach to assay transmembrane proteins on exosomes. While high-copy-number proteins could be readily detected, detecting low-copy-number proteins was improved using a lipophilic tracer that clustered exosomes. To illustrate this, we observed that exosomes derived from SKBR3 cells, a cell model for human HER2+ breast cancer, contained both HER1 and HER2 but at different levels of abundance. Collectively, these new methods will help to ensure a consistent framework to identify specific roles that exosomes play in regulating cell-to-cell communication.
作为一种分泌性膜泡，外泌体是一种新兴的细胞间通讯方式。然而，由于外泌体样本通常会被其他细胞外囊泡污染，外泌体在调节免疫和促进肿瘤发生中的生物学作用仍存在争议。我们怀疑现有方法是否会扭曲我们对外泌体生物学的认识，因此比较了两种直接成像细胞外囊泡的方法，并量化了不同生产和储存条件对外泌体样本质量的影响。将扫描电子显微镜（SEM）与透射电子显微镜（TEM）进行比较，作为检查外泌体形态的替代方法。使用SEM，我们能够根据大小分布将外泌体与其他污染性细胞外囊泡区分开来。更重要的是，在进行SEM成像之前对样本进行冷冻，使得更难以将外泌体与细胞死亡期间分泌的细胞外囊泡区分开来。除了形态学，还对外泌体内所含RNA在不同储存条件下的质量进行了表征，样本冷冻也会使RNA降解。最后，我们开发了一种新的流式细胞术方法来检测外泌体上的跨膜蛋白。虽然可以很容易地检测到高拷贝数的蛋白，但使用聚集外泌体的亲脂性示踪剂可以改善低拷贝数蛋白的检测。为了说明这一点，我们观察到源自人HER2 +乳腺癌细胞模型SKBR3细胞的外泌体同时含有HER1和HER2，但丰度水平不同。总的来说，这些新方法将有助于确保一个一致的框架，以确定外泌体在调节细胞间通讯中所起的特定作用。
