title: Heparin affinity purification of extracellular vesicles.
abstract: Extracellular vesicles (EVs) are lipid membrane vesicles released by cells. They carry active biomolecules including DNA, RNA, and protein which can be transferred to recipient cells. Isolation and purification of EVs from culture cell media and biofluids is still a major challenge. The most widely used isolation method is ultracentrifugation (UC) which requires expensive equipment and only partially purifies EVs. Previously we have shown that heparin blocks EV uptake in cells, supporting a direct EV-heparin interaction. Here we show that EVs can be purified from cell culture media and human plasma using ultrafiltration (UF) followed by heparin-affinity beads. UF/heparin-purified EVs from cell culture displayed the EV marker Alix, contained a diverse RNA profile, had lower levels of protein contamination, and were functional at binding to and uptake into cells. RNA yield was similar for EVs isolated by UC. We were able to detect mRNAs in plasma samples with comparable levels to UC samples. In conclusion, we have discovered a simple, scalable, and effective method to purify EVs taking advantage of their heparin affinity.
authors.affiliation: Department of Neurology, Massachusetts General Hospital and Program in Neuroscience, Harvard Medical School, Boston, MA.
authors.forename: Leonora
authors.initials: L
authors.lastname: Balaj
authors.affiliation: 1] Department of Neurology, Massachusetts General Hospital and Program in Neuroscience, Harvard Medical School, Boston, MA. [2] Department of Cell Biology and Histology, Academic Medical Center (AMC), University of Amsterdam, The Netherlands.
authors.forename: Nadia A
authors.initials: NA
authors.lastname: Atai
authors.affiliation: Department of Neurology, Massachusetts General Hospital and Program in Neuroscience, Harvard Medical School, Boston, MA.
authors.forename: Weilin
authors.initials: W
authors.lastname: Chen
authors.affiliation: Department of Neurology, Massachusetts General Hospital and Program in Neuroscience, Harvard Medical School, Boston, MA.
authors.forename: Dakai
authors.initials: D
authors.lastname: Mu
authors.affiliation: Department of Neurology, Massachusetts General Hospital and Program in Neuroscience, Harvard Medical School, Boston, MA.
authors.forename: Bakhos A
authors.initials: BA
authors.lastname: Tannous
authors.affiliation: 1] Department of Neurology, Massachusetts General Hospital and Program in Neuroscience, Harvard Medical School, Boston, MA. [2] Departments of Radiology, Massachusetts General Hospital and Program in Neuroscience, Harvard Medical School, Boston, MA.
authors.forename: Xandra O
authors.initials: XO
authors.lastname: Breakefield
authors.affiliation: Exosome Diagnostics Inc. Cambridge, MA.
authors.forename: Johan
authors.initials: J
authors.lastname: Skog
authors.affiliation: Department of Neurology, Massachusetts General Hospital and Program in Neuroscience, Harvard Medical School, Boston, MA.
authors.forename: Casey A
authors.initials: CA
authors.lastname: Maguire
mesh_terms: D045744:Cell Line, Tumor; D000067128:Extracellular Vesicles / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D008863:Microspheres; D011485:Protein Binding / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D012685:Sepharose / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; D006493:Heparin; D012685:Sepharose