Cushioned-Density Gradient Ultracentrifugation (C-DGUC): A Refined and High Performance Method for the Isolation, Characterization, and Use of Exosomes.
Cushioned-Density Gradient Ultracentrifugation (C-DGUC): A Refined and High Performance Method for the Isolation, Characterization, and Use of Exosomes.
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缓冲密度梯度超速离心（C-DGUC）：一种用于隔离，表征和使用外泌体的精制和高性能方法。
DOI：
10.1007/978-1-4939-7652-2_7
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Raffai RL
中科院分区：
文献类型：
--
作者：
Li K;Wong DK;Hong KY;Raffai RL
外泌体代表一类细胞外囊泡，被认为在所有细胞类型中脱落。尽管外泌体生物发生和功能的确切性质仍不完全清楚，但它们越来越被认为是健康和疾病中细胞间通讯的来源。最近观察到通过供体细胞衍生的外泌体在受体细胞中发挥遗传调控作用的RNA交换导致了外泌体研究的福音。外泌体作为一种新的人类病理治疗途径的兴奋和前景仍然受到与它们从培养基和生物液体中分离相关的挑战的限制。引入新的方法来促进外泌体的分离，同时也引起了对描述外泌体效应功能的研究的可重复性的关注。即使是高速超离心，第一种长期以来被认为是外泌体分离的金标准方法，最近也被注意到受到可能影响外泌体制备功能读数的不受控制的变量的影响。本章描述了通过首先将外泌体浓缩在液体缓冲液中，然后使用密度梯度超离心来解决这些限制的原理和方法。我们的方法避免了直接在塑料管上制造颗粒可能带来的并发症，并允许从致密蛋白聚集体中进一步纯化外泌体。
Exosomes represent one class of extracellular vesicles that are thought to be shed by all cell types. Although the exact nature of exosome biogenesis and function remains incompletely understood, they are increasingly recognized as a source of intercellular communication in health and disease. Recent observations of RNA exchange via donor cell-derived exosomes that exert genetic regulation in recipient cells have led to a boon into exosome research. The excitement and promise of exosomes as a new therapeutic avenue for human pathologies remain limited by challenges associated with their isolation from culture media and bio-fluids. The introduction of new methodologies to facilitate the isolation of exosomes has simultaneously raised concerns related to the reproducibility of studies describing exosome effector functions. Even high speed ultracentrifugation, the first and long considered gold standard approach for exosome isolation has recently been noted to be subject to uncontrolled variables that could impact functional readouts of exosome preparations. This chapter describes principles and methods that attempt to overcome such limitations by first concentrating exosomes in a liquid cushion and subsequently resolving them using density gradient ultracentrifugation. Our approach avoids possible complications associated with direct pelleting onto plastic tubes and allows for further purification of exosomes from dense protein aggregates.