Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium.
Cancer Research Institute Ghent, Ghent, Belgium.
J Nanobiotechnology. 2023 May 19;21(1):157. doi: 10.1186/s12951-023-01917-z.
Extracellular vesicles (EV) are extensively studied in human body fluids as potential biomarkers for numerous diseases. Major impediments of EV-based biomarker discovery include the specificity and reproducibility of EV sample preparation as well as intensive manual labor. We present an automated liquid handling workstation for the density-based separation of EV from human body fluids and compare its performance to manual handling by (in)experienced researchers.
Automated versus manual density-based separation of trackable recombinant extracellular vesicles (rEV) spiked in PBS significantly reduces variability in rEV recovery as quantified by fluorescent nanoparticle tracking analysis and ELISA. To validate automated density-based EV separation from complex body fluids, including blood plasma and urine, we assess reproducibility, recovery, and specificity by mass spectrometry-based proteomics and transmission electron microscopy. Method reproducibility is the highest in the automated procedure independent of the matrix used. While retaining (in urine) or enhancing (in plasma) EV recovery compared to manual liquid handling, automation significantly reduces the presence of body fluid specific abundant proteins in EV preparations, including apolipoproteins in plasma and Tamm-Horsfall protein in urine.
In conclusion, automated liquid handling ensures cost-effective EV separation from human body fluids with high reproducibility, specificity, and reduced hands-on time with the potential to enable larger-scale biomarker studies.
细胞外囊泡（EV）在人体体液中作为许多疾病的潜在生物标志物得到了广泛研究。基于 EV 的生物标志物发现的主要障碍包括 EV 样本制备的特异性和重现性以及大量的人工劳动。我们提出了一种自动化液体处理工作站，用于基于密度从人体体液中分离 EV，并将其性能与经验丰富和缺乏经验的研究人员的手动处理进行比较。
自动化与手动基于密度的分离可显着降低荧光纳米颗粒跟踪分析和 ELISA 定量的追踪可重构细胞外囊泡（rEV）在 PBS 中回收的变异性。为了验证从复杂体液（包括血浆和尿液）中自动化基于密度的 EV 分离，我们通过基于质谱的蛋白质组学和透射电子显微镜评估重现性、回收率和特异性。自动化程序的方法重现性最高，与使用的基质无关。与手动液体处理相比，自动化不仅保留了（在尿液中）或提高了（在血浆中）EV 的回收率，而且还显着降低了 EV 制剂中体液特异性丰富蛋白的存在，包括血浆中的载脂蛋白和尿液中的 Tamm-Horsfall 蛋白。
总之，自动化液体处理可确保从人体体液中以高重现性、特异性和减少人工干预的成本效益方式分离 EV，从而有可能实现更大规模的生物标志物研究。
