Isolating Small Extracellular Vesicles from Small Volumes of Blood Plasma using size exclusion chromatography and density gradient ultracentrifugation: A Comparative Study.
Isolating Small Extracellular Vesicles from Small Volumes of Blood Plasma using size exclusion chromatography and density gradient ultracentrifugation: A Comparative Study.
复制标题
使用尺寸排阻色谱法和密度梯度超速离心从小体积血浆中分离小细胞外囊泡：一项比较研究。
DOI：
10.1101/2023.10.30.564707
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Dao,Ming
中科院分区：
文献类型：
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作者：
Kong,Fang;Upadya,Megha;Wong,AndrewSeeWeng;Dalan,Rinkoo;Dao,Ming
细胞外小泡（Small extracellular vesicles，sEVs）是细胞在生理和病理条件下释放的异质性生物囊泡。由于它们作为人类血液中有价值的诊断和预后生物标志物的潜力，迫切需要开发用于从含有丰富血浆蛋白和脂蛋白的复杂血浆环境中分离高纯度sEV的有效方法。尺寸排阻色谱法（SEC）和密度梯度超离心（DGUC）是两种常用的分离技术，已显示出解决这一挑战的前景。在这项研究中，我们的目的是确定SEC和DGUC的最佳组合和顺序，用于从小血浆体积中分离sEV，以提高所得分离株的效率和纯度。为了实现这一点，我们比较了使用两种组合的sEV分离：SEC-DGUC和DGUC-SEC，从500 μl血浆的单位体积。两种方案都成功分离了高纯度的sEV;然而，SEC-DGUC组合产生了更高的sEV蛋白和RNA含量。我们使用流式细胞术和质谱法进一步表征了从SEC-DGUC方案获得的分离的sEV，以评估其质量和纯度。总之，优化的SEC-DGUC方案是有效的，高度可重复的，并且非常适合从小血容量中分离高纯度sEV。
Small extracellular vesicles (sEVs) are heterogeneous biological vesicles released by cells under both physiological and pathological conditions. Due to their potential as valuable diagnostic and prognostic biomarkers in human blood, there is a pressing need to develop effective methods for isolating high-purity sEVs from the complex milieu of blood plasma, which contains abundant plasma proteins and lipoproteins. Size exclusion chromatography (SEC) and density gradient ultracentrifugation (DGUC) are two commonly employed isolation techniques that have shown promise in addressing this challenge. In this study, we aimed to determine the optimal combination and sequence of SEC and DGUC for isolating sEVs from small plasma volumes, in order to enhance both the efficiency and purity of the resulting isolates. To achieve this, we compared sEV isolation using two combinations: SEC-DGUC and DGUC-SEC, from unit volumes of 500 μl plasma. Both protocols successfully isolated high-purity sEVs; however, the SEC-DGUC combination yielded higher sEV protein and RNA content. We further characterized the isolated sEVs obtained from the SEC-DGUC protocol using flow cytometry and mass spectrometry to assess their quality and purity. In conclusion, the optimized SEC-DGUC protocol is efficient, highly reproducible, and well-suited for isolating high-purity sEVs from small blood volumes.