Zoia Matteo, Yesodha Subramanian Bibin, Eriksson Klara Kristin, Ravi Meera Sruthi, Yaghmaei Shekoofeh, Fellay Isabelle, Scolari Brigitte, Walch Michael, Mantel Pierre-Yves
Faculty of Science and Medicine, Department of Oncology, Microbiology and Immunology, Anatomy Unit, University of Fribourg, Fribourg, Switzerland.
Department for BioMedical Research (DBMR), University of Bern, Bern, Switzerland.
Front Cell Dev Biol. 2022 May 16;10:812244. doi: 10.3389/fcell.2022.812244. eCollection 2022.
Malaria affects the poorer regions of the world and is of tremendous health and economic burden for developing countries. Extracellular vesicles (EVs) are small vesicles released by almost any cells in the human body, including malaria infected red blood cells. Recent evidence shows that EVs might contribute to the pathogenesis of malaria. In addition, EVs hold considerable value in biomarker discovery. However, there are still significant gaps in our understanding of EV biology. So far most of our knowledge about EVs in malaria comes from work. More field studies are required to gain insight into their contribution to the disease and pathogenesis under physiological conditions. However, to perform research on EVs in low-income regions might be challenging due to the lack of appropriate equipment to isolate EVs. Therefore, there is a need to develop and validate EV extraction protocols applicable to poorly equipped laboratories. We established and validated two protocols for EV isolation from cell culture supernatants, rodent and human plasma. We compared polyethylene glycol (PEG) and salting out (SA) with sodium acetate for precipitation of EVs. We then characterized the EVs by Transmission Electron Microscopy (TEM), Western Blot, Size-exclusion chromatography (SEC), bead-based flow cytometry and protein quantification. Both protocols resulted in efficient purification of EVs without the need of expensive material or ultracentrifugation. Furthermore, the procedure is easily scalable to work with large and small sample volumes. Here, we propose that both of our approaches can be used in resource limited countries, therefore further helping to close the gap in knowledge of EVs during malaria.
疟疾影响着世界上较为贫困的地区，给发展中国家带来了巨大的健康和经济负担。细胞外囊泡（EVs）是人体几乎所有细胞释放的小囊泡，包括感染疟疾的红细胞。最近的证据表明，EVs可能在疟疾发病机制中起作用。此外，EVs在生物标志物发现方面具有重要价值。然而，我们对EV生物学的理解仍存在重大差距。到目前为止，我们对疟疾中EVs的了解大多来自相关研究。需要更多的实地研究来深入了解它们在生理条件下对疾病和发病机制的作用。然而，由于缺乏分离EVs的适当设备，在低收入地区开展EVs研究可能具有挑战性。因此，有必要开发和验证适用于设备简陋实验室的EV提取方案。我们建立并验证了两种从细胞培养上清液、啮齿动物和人类血浆中分离EVs的方案。我们将聚乙二醇（PEG）和盐析法（SA）与醋酸钠用于EVs的沉淀进行了比较。然后，我们通过透射电子显微镜（TEM）、蛋白质印迹、尺寸排阻色谱（SEC）、基于微珠的流式细胞术和蛋白质定量对EVs进行了表征。两种方案都能有效纯化EVs，无需昂贵的材料或超速离心。此外，该方法易于扩展以处理大体积和小体积样本。在此，我们建议我们的两种方法都可用于资源有限的国家，从而进一步有助于缩小疟疾期间对EVs认识上的差距。
