Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA.
Nancy E. and Peter C. Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA.
J Struct Biol. 2020 Apr 1;210(1):107474. doi: 10.1016/j.jsb.2020.107474. Epub 2020 Feb 4.
As interest in the role of extracellular vesicles in cell-to-cell communication has increased, so has the use of microscopy and analytical techniques to assess their formation, release, and morphology. In this study, we evaluate scanning electron microscopy (SEM) and cryo-SEM for characterizing the formation and shedding of vesicles from human breast cell lines, parental and hyaluronan synthase 3-(HAS3)-overexpressing MCF10A cells, grown directly on transmission electron microscopy (TEM) grids. While cells imaged with conventional and cryo-SEM exhibit distinct morphologies due to the sample preparation process for each technique, tubular structures protruding from the cell surfaces were observed with both approaches. For HAS3-MCF10A cells, vesicles were present along the length of membrane protrusions. Once completely shed from the cells, extracellular vesicles were characterized using nanoparticle tracking analysis (NTA) and cryo-TEM. The size distributions obtained by each technique were different not only in the range of vesicles analyzed, but also in the relative proportion of smaller-to-larger vesicles. These differences are attributed to the presence of biological debris in the media, which is difficult to differentiate from vesicles in NTA. Furthermore, we demonstrate that cryo-TEM can be used to distinguish between vesicles based on their respective surface structures, thereby providing a path to differentiating vesicle subpopulations and identifying their size distributions. Our study emphasizes the necessity of pairing several techniques to characterize extracellular vesicles.
随着人们对细胞间通讯中外泌体作用的兴趣日益增加，用于评估其形成、释放和形态的显微镜和分析技术的使用也越来越多。在这项研究中，我们评估了扫描电子显微镜（SEM）和冷冻扫描电子显微镜（cryo-SEM）在直接在透射电子显微镜（TEM）网格上培养的人乳腺细胞系、亲本和透明质酸合酶 3（HAS3）过表达 MCF10A 细胞中用于表征囊泡形成和脱落的能力。虽然用常规和 cryo-SEM 成像的细胞由于每种技术的样品制备过程而表现出不同的形态，但用这两种方法都观察到了从细胞表面突出的管状结构。对于 HAS3-MCF10A 细胞，囊泡存在于膜突起的长度上。一旦完全从细胞中脱落，就使用纳米颗粒跟踪分析（NTA）和冷冻 TEM 对细胞外囊泡进行表征。两种技术获得的粒径分布不仅在分析的囊泡范围内不同，而且在较小到较大囊泡的相对比例上也不同。这些差异归因于存在于介质中的生物碎片，这在 NTA 中很难与囊泡区分开来。此外，我们证明 cryo-TEM 可用于根据其各自的表面结构区分囊泡，从而为区分囊泡亚群和识别其粒径分布提供了一种途径。我们的研究强调了需要结合几种技术来表征细胞外囊泡。
