Current analysis of exosomes focuses primarily on bulk analysis, where exosome-to-exosome variability cannot be assessed. In this study, we used Raman spectroscopy to study the chemical composition of single exosomes. We measured spectra of individual exosomes from 8 cell lines. Cell-line-averaged spectra varied considerably, reflecting the variation in total exosomal protein, lipid, genetic, and cytosolic content. Unexpectedly, single exosomes isolated from the same cell type also exhibited high spectral variability. Subsequent spectral analysis revealed clustering of single exosomes into 4 distinct groups that were not cell-line specific. Each group contained exosomes from multiple cell lines, and most cell lines had exosomes in multiple groups. The differences between these groups are related to chemical differences primarily due to differing membrane composition. Through a principal components analysis, we identified that the major sources of spectral variation among the exosomes were in cholesterol content, relative expression of phospholipids to cholesterol, and surface protein expression. For example, exosomes derived from cancerous versus non-cancerous cell lines can be largely separated based on their relative expression of cholesterol and phospholipids. We are the first to indicate that exosome subpopulations are shared among cell types, suggesting distributed exosome functionality. The origins of these differences are likely related to the specific role of extracellular vesicle subpopulations in both normal cell function and carcinogenesis, and they may provide diagnostic potential at the single exosome level.

目前对外泌体的分析主要集中于大量分析，其中无法评估外泌体之间的变异性。在这项研究中，我们使用拉曼光谱研究单个外泌体的化学成分。我们测量了来自8个细胞系的单个外泌体的光谱。细胞系平均光谱变化很大，反映出总外泌体蛋白质，脂质，遗传和胞浆含量的变化。出乎意料的是，从相同细胞类型中分离出的单个外泌体也表现出高光谱变异性。随后的光谱分析显示单个外泌体聚类为不是细胞系特异性的4个不同的组。每组包含来自多个细胞系的外泌体，大多数细胞系在多个组中具有外泌体。这些组之间的差异主要与化学差异有关，这主要是由于膜组成不同所致。通过主成分分析，我们确定了外泌体之间光谱变化的主要来源是胆固醇含量，磷脂相对于胆固醇的相对表达以及表面蛋白表达。例如，基于癌细胞和非癌细胞系的外泌体可根据其胆固醇和磷脂的相对表达而大大分离。我们是第一个指出外泌体亚群在细胞类型之间共享的人，这暗示了外泌体的功能性分布。这些差异的起源可能与细胞外囊泡亚群在正常细胞功能和致癌性中的特定作用有关，