ABSTRACT

Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque, and mouse. Effects of tissue perfusion and a model of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with those of macaque and, especially, mouse, suggesting opportunities for optimization. Where possible, perfusion should be performed in animal models. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Finally, different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease.

摘要

细胞外囊泡（EVs）通过将物质穿梭到细胞外和细胞间而参与广泛的生理和病理过程。因此，组织电动车在释放到易于获取的生物体液中时，可能有助于深入了解疾病机理，还可以出卖疾病。由于脑源电动车（bdEV）及其货物可能是神经退行性疾病的生物标记，因此我们评估了对已发表的严格的从脑组织中分离电动车的协议的修改，并研究了加工变量对定量和定性结果的影响。为此，将尺寸排阻色谱法（SEC）和蔗糖密度梯度超速离心作为涉及逐步超速离心的方案中的最终分离步骤进行了比较。bdEV与人，猕猴和小鼠的脑组织分离。探讨了最终bdEV分离之前的组织灌注效果和验尸间隔（PMI）模型。进行了符合MISEV2018的EV表征，并完成了小RNA和蛋白质谱分析。我们得出的结论是，采用SEC的改进协议可实现EV分离效率，与使用梯度密度超速离心的协议大致相似，同时减少了操作员时间，并可能减少了可变性。与猕猴（尤其是小鼠）相比，该协议似乎可为人类组织产生更高纯度的bdEV。如有可能，应在动物模型中进行灌注。死亡/组织存储/处理与最终bdEV分离之间的间隔也会影响bdEV的种群和组成，因此应记录下来以进行严格报告。最后，通过本文报道的改良方法获得的不同的EV群体显示出暗示生物标志物潜力的特征性RNA和蛋白质含量。总而言之，这项研究发现，SEC的可自动化和日益广泛使用的技术可以应用于组织EV分离，并且还揭示了更多有关使用组织EV时特定物种和技术考虑因素的重要性。这些结果有望增强bdEV在揭示和了解脑部疾病中的应用。并进一步揭示了与组织电动车一起使用时特定物种和技术考虑因素的重要性。这些结果有望增强bdEV在揭示和了解脑部疾病中的应用。并进一步揭示了与组织电动车一起使用时特定物种和技术考虑因素的重要性。这些结果有望增强bdEV在揭示和了解脑部疾病中的应用。