Multimode chromatography‐based techniques for high purity isolation of extracellular vesicles from human blood plasma

Abstract Extracellular vesicles (EVs) play a pivotal role in various biological pathways, such as immune responses and the progression of diseases, including cancer. However, it is challenging to isolate EVs at high purity from blood plasma and other biofluids due to their low abundance compared to more predominant biomolecular species such as lipoprotein particles and free protein complexes. Ultracentrifugation‐based EV isolation, the current gold standard technique, cannot overcome this challenge due to the similar biophysical characteristics of such species. We developed several novel approaches to enrich EVs from plasma while depleting contaminating molecular species using multimode chromatography‐based strategies. On average, we identified 716 ± 68 and 1054 ± 35 protein groups in EV isolates from 100 µL of plasma using multimode chromatography‐ and ultracentrifugation‐based techniques, respectively. The developed methods resulted in similar EV isolates purity, providing significant advantages in simplicity, throughput, scalability, and applicability for various downstream analytical and potential clinical applications

Human red blood cells release microvesicles with distinct sizes and protein composition that alter neutrophil phagocytosis

Abstract Extracellular vesicles (EVs) are membrane‐bound structures released by cells and tissues into biofluids, involved in cell‐cell communication. In humans, circulating red blood cells (RBCs), represent the most common cell‐type in the body, generating daily large numbers of microvesicles. In vitro, RBC vesiculation can be mimicked by stimulating RBCs with calcium ionophores, such as ionomycin and A23187. The fate of microvesicles released during in vivo aging of RBCs and their interactions with circulating cells is hitherto unknown. Using SEC plus DEG isolation methods, we have found that human RBCs generate microvesicles with two distinct sizes, densities and protein composition, identified by flow cytometry, and MRPS, and further validated by immune TEM. Furthermore, proteomic analysis revealed that RBC‐derived microvesicles (RBC‐MVs) are enriched in proteins with important functions in ion channel regulation, calcium homeostasis and vesicular transport, such as of sorcin, stomatin, annexin A7 and RAB proteins. Cryo‐electron microscopy identified two separate pathways of RBC‐MV‐neutrophil interaction, direct fusion with the plasma membrane and internalization, respectively. Functionally, RBC‐MVs decrease neutrophil ability to phagocytose Escherichia coli but do not affect their survival at 24 h. This work brings new insights regarding the complexity of the RBC‐MVs biogenesis, as well as their possible role in circulation