Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues
Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues
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DOI：
10.3390/ijms21155586
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发表时间：
2020-08-01
影响因子：
5.6
通讯作者：
Seiffert, Martina
中科院分区：
文献类型：
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作者：
Bordas, Marie;Genard, Geraldine;Seiffert, Martina
小细胞外囊泡（sEV）是负责由健康细胞和癌细胞释放的细胞与细胞通信的纳米颗粒。已经描述了sEV在生理和病理背景下的不同作用，包括加速组织再生，调节肿瘤微环境或转移前生态位形成，并且它们被讨论为体液中诊断和预后的有前途的生物标志物。尽管已经努力使sEV的分离和表征技术标准化，但目前的方案通常导致可溶性蛋白质或脂质复合物和其他细胞外囊泡的共分离。当从组织中分离sEV时，受污染制剂的风险特别高。因此，旨在理解sEV功能作用的数据解释仍然具有挑战性和不一致性。在这里，我们报告了一个优化的协议，从人类和小鼠淋巴组织分离的sEV。从新鲜切除的人淋巴结和鼠脾分离sEV，比较两种不同的方法-（1）在蔗糖密度垫上的超离心和（2）组合的超离心与尺寸排阻色谱。使用最先进的技术分析sEV制剂的纯度，包括免疫印迹、纳米颗粒跟踪分析和电子显微镜。我们的结果清楚地表明了尺寸排阻色谱法的优越性，这导致了sEV的更高的产率和纯度，并且我们表明它们的功能在两种分离方案之间显着改变。
Small extracellular vesicles (sEVs) are nanoparticles responsible for cell-to-cell communication released by healthy and cancer cells. Different roles have been described for sEVs in physiological and pathological contexts, including acceleration of tissue regeneration, modulation of tumor microenvironment, or premetastatic niche formation, and they are discussed as promising biomarkers for diagnosis and prognosis in body fluids. Although efforts have been made to standardize techniques for isolation and characterization of sEVs, current protocols often result in co-isolation of soluble protein or lipid complexes and of other extracellular vesicles. The risk of contaminated preparations is particularly high when isolating sEVs from tissues. As a consequence, the interpretation of data aiming at understanding the functional role of sEVs remains challenging and inconsistent. Here, we report an optimized protocol for isolation of sEVs from human and murine lymphoid tissues. sEVs from freshly resected human lymph nodes and murine spleens were isolated comparing two different approaches-(1) ultracentrifugation on a sucrose density cushion and (2) combined ultracentrifugation with size-exclusion chromatography. The purity of sEV preparations was analyzed using state-of-the-art techniques, including immunoblots, nanoparticle tracking analysis, and electron microscopy. Our results clearly demonstrate the superiority of size-exclusion chromatography, which resulted in a higher yield and purity of sEVs, and we show that their functionality alters significantly between the two isolation protocols.