Division of Neuroscience, Institute of Experimental Neurology, San Raffaele Scientific Institute, Via Olgettina, 60, 20132, Milan, Italy.
Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy.
Sci Rep. 2021 Feb 11;11(1):3589. doi: 10.1038/s41598-021-83241-w.
Microvesicles (MVs) are large extracellular vesicles differing in size, cargo and composition that share a common mechanism of release from the cells through the direct outward budding of the plasma membrane. They are involved in a variety of physiological and pathological conditions and represent promising biomarkers for diseases. MV heterogeneity together with the lack of specific markers had strongly hampered the development of effective methods for MV isolation and differential centrifugation remains the most used method to purify MVs. In this study, we analysed the capacity of the differential centrifugation method to isolate MVs from cell-conditioned medium using flow cytometry and TEM/AFM microscopy. We found that the loss of MVs (general population and/or specific subpopulations) represents a major and underestimate drawback of the differential centrifugation protocol. We demonstrate that the choice of the appropriate rotor type (fixed-angle vs swinging-bucket) and the implementation of an additional washing procedure to the first low-speed centrifugation step of the protocol allow to overcome this problem increasing the total amount of isolated vesicles and avoiding the selective loss of MV subpopulations. These parameters/procedures should be routinely employed into optimized differential centrifugation protocols to ensure isolation of the high-quantity/quality MVs for the downstream analysis/applications.
微泡（MVs）是大小、货物和成分不同的大型细胞外囊泡，通过细胞质膜的直接向外出芽，具有共同的释放机制。它们参与多种生理和病理状况，并代表疾病有希望的生物标志物。MV 的异质性以及缺乏特异性标记物强烈阻碍了有效的 MV 分离方法的发展，差速离心仍然是纯化 MV 的最常用方法。在这项研究中，我们使用流式细胞术和 TEM/AFM 显微镜分析了差速离心法从细胞条件培养基中分离 MV 的能力。我们发现，MV（一般群体和/或特定亚群）的丢失是差速离心方案的一个主要和低估的缺点。我们证明，选择适当的转子类型（定角 vs 摆动桶）并在协议的第一步低速离心步骤中实施额外的洗涤程序，可以克服这个问题，增加分离囊泡的总量，并避免 MV 亚群的选择性丢失。这些参数/程序应常规应用于优化的差速离心方案中，以确保下游分析/应用中分离出高质量/数量的 MV。
