Balancing yield, purity and practicality: a modified differential ultracentrifugation protocol for efficient isolation of small extracellular vesicles from human serum

Ultracentrifugation remains the gold standard for isolation of small extracellular vesicles (sEV), particularly for cancer applications. The objective of this study was to determine if a widely used ultracentrifugation protocol for isolation of serum sEV could be modified to reduce the number of ultracentrifugation cycles and increase efficiency, while maintaining equal or better sample purity and yield. Serum was obtained from two healthy subjects. sEVs were isolated from 1 mL aliquots using three different ultracentrifugation protocols. Co-isolation of RNA carrier protein was assessed by performing Western blots for ApoA-I, ApoB, and Ago2. Small RNA-sequencing was performed on the sEV isolates, and differential detection of small ncRNA was compared across isolation protocols. Reduction from three- to two-ultracentrifuge cycles with no sucrose cushion resulted in a much higher sEV yield but also had the highest levels of lipoprotein and Ago2 contamination. However, the two-ultracentrifugation cycle protocol that incorporated a 30% sucrose cushion into the first cycle resulted in slightly higher sEV yields with lower levels of protein contamination compared to the lengthier three-ultracentrifugation cycle approach, therefore presenting a more efficient alternative approach for isolation of serum sEVs. It was also notable that there were some differences in sEV ncRNA cargo according to protocol, although it was less than expected given the differences in co-isolated RNA carrier proteins. Our results suggest that use of the modified serum sEV isolation protocol with two ultracentrifugation cycles and incorporating a 30% sucrose cushion offers a more efficient approach in terms of efficiency and purity.

超速离心法（ultracentrifugation）仍是分离小型细胞外囊泡（small extracellular vesicles, sEV）的金标准，在癌症相关研究领域应用尤为广泛。本研究旨在优化一套广泛使用的血清sEV分离超速离心方案，在保证样本纯度与提取量不低于甚至优于原方案的前提下，减少超速离心循环次数并提升分离效率。本研究从2名健康受试者体内采集血清，针对1mL等分血清样本，采用3种不同的超速离心方案分离sEV。通过对ApoA-I、ApoB及Ago2蛋白进行蛋白质免疫印迹实验（Western blot），评估共分离的RNA载体蛋白水平。对分离得到的sEV进行小RNA测序，对比不同分离方案下小型非编码RNA（small ncRNA）的差异检出情况。结果显示，省略蔗糖垫层、将超速离心循环从3次缩减至2次时，sEV提取量显著提升，但脂蛋白与Ago2蛋白污染水平也达到最高。不过，在首轮超速离心步骤中加入30%蔗糖垫层的2次循环离心方案，相较于耗时更长的3次循环离心方案，虽sEV提取量仅略有提升，但蛋白污染水平更低，因此可作为血清sEV分离的更高效替代方案。值得注意的是，不同分离方案得到的sEV非编码RNA载荷存在一定差异，尽管相较于共分离RNA载体蛋白的差异而言，该差异小于预期。本研究结果表明，采用含2次超速离心循环并在首轮加入30%蔗糖垫层的优化方案，可在分离效率与样本纯度两方面实现更优的血清sEV分离效果。