Optimized exosome isolation protocol for cell culture supernatant and human plasma.
Optimized exosome isolation protocol for cell culture supernatant and human plasma.
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DOI：
10.3402/jev.v4.27031
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发表时间：
2015
影响因子：
16
通讯作者：
Möller A
中科院分区：
文献类型：
--
作者：
Lobb RJ;Becker M;Wen SW;Wong CS;Wiegmans AP;Leimgruber A;Möller A
细胞外囊泡是疾病诊断和预后中新型生物标志物的丰富来源。然而，目前有有限的信息阐明了从细胞培养上清液和复杂的生物流体如血浆中获得高产量的纯外泌体（细胞外囊泡的子集）的最有效方法。为此，我们全面表征了各种外泌体分离方案的效率、产率和分离的外泌体的纯度。重复的超离心步骤可降低外泌体制备物的质量，导致较低的外泌体产率。我们表明，使用超滤装置的细胞培养条件培养基的浓度与传统的超滤方案相比，导致囊泡分离增加。然而，我们关于使用从非小细胞肺癌（NSCLC）SK-MES-1细胞系分离的条件培养基的数据表明，浓缩装置的选择可以极大地影响分离的外泌体的产量。我们发现基于离心的浓缩方法比压力驱动的浓缩装置更合适，并且允许从NSCLC细胞培养条件培养基和复杂生物液体中快速分离外泌体。事实上，迄今为止，还没有详细描述利用当前商业方法从细胞和患者样品分离外泌体的方案。利用可调电阻脉冲传感和蛋白质分析，我们提供了4个外泌体分离技术的比较分析，表明其效率和制备纯度。我们的结果表明，目前用于从细胞培养条件培养基和血浆中分离外泌体的沉淀方案提供了最不纯的外泌体制备物，而尺寸排阻分离与外泌体的密度梯度纯化相当。我们已经确定了目前常见的细胞外囊泡分离方法的缺点，并提供了一个潜在的标准化方法，是有效的，可重复的，可用于各种起始材料。我们相信这种方法将在日益增长的细胞外囊泡研究领域中有广泛的应用。
Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research.