Wierz Marina, Pierson Sandrine, Gargiulo Ernesto, Guerin Coralie, Moussay Etienne, Paggetti Jerome
Laboratory of Experimental Cancer Research, Department of Oncology, Luxembourg Institute of Health, Luxembourg, Luxembourg.
National Cytometry Platform, Department of Infection and Immunity, Luxembourg Institute of Health, Luxembourg, Luxembourg.
Methods Mol Biol. 2019;1884:231-245. doi: 10.1007/978-1-4939-8885-3_16.
Exosomes are membrane-enclosed vesicles released by different cell types into the extracellular space. As mediators of intercellular communication, they are involved in multiple physiological processes, but they are also associated with the pathogenesis of human malignancies including leukemia. Isolation of exosomes enables the characterization of their role in microenvironment modulation as well as their participation in disease pathology. A variety of strategies and techniques exists to purify exosomes from many biological fluids (e.g., blood, urine, and saliva). Here, we describe the efficient production of large quantities of exosomes from leukemic cell lines by using CELLine bioreactors based on two-compartment technology, as well as their isolation and purification by combining differential centrifugation and ultracentrifugation through a density gradient (17% OptiPrep cushion). Thus, exosomes are appropriately prepared for characterization by western blotting to detect exosome markers or imaging flow cytometry (ImageStream), and for downstream analyses such as the internalization in microenvironmental cells by confocal imaging or flow cytometry, methods which are also described in this chapter.
外泌体是由不同细胞类型释放到细胞外空间的膜包被囊泡。作为细胞间通讯的介质，它们参与多种生理过程，但也与包括白血病在内的人类恶性肿瘤的发病机制有关。外泌体的分离有助于表征其在微环境调节中的作用以及它们在疾病病理学中的参与情况。存在多种策略和技术可从许多生物流体（例如血液、尿液和唾液）中纯化外泌体。在这里，我们描述了通过使用基于两室技术的CELLine生物反应器从白血病细胞系中高效生产大量外泌体，以及通过差速离心和通过密度梯度（17% OptiPrep垫层）的超速离心相结合来分离和纯化它们。因此，外泌体经过适当制备，可通过蛋白质印迹检测外泌体标志物或成像流式细胞术（ImageStream）进行表征，并用于下游分析，例如通过共聚焦成像或流式细胞术分析微环境细胞中的内化情况，本章也介绍了这些方法。
