A NOVEL AFFINITY-BASED METHOD FOR EXOSOME ISOLATION ISOLATION OF HIGHLY PURIFIED AND INTACT EXOSOMES AND OTHER EXTRACELLULAR VESICLES

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1 INNOVACIÓN TECNOLÓGICA PARA LABORATORIO A NOVEL AFFINITY-BASED METHOD FOR EXOSOME ISOLATION ISOLATION OF HIGHLY PURIFIED AND INTACT EXOSOMES AND OTHER EXTRACELLULAR VESICLES

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3 A NOVEL AFFINITY-BASED METHOD FOR EXOSOME ISOLATION INTRODUCTION Exosomes are small extracellular vesicles ( nm in diameter) carrying mrna, mirna, DNA as well as various secretory proteins. [1-3] They are ubiquitous components of body fluids and can be found in blood, saliva, urine, cerebrospinal fluid (CSF) and breast milk. [4] Although their exact biological function is not yet fully characterized, increasing evidence is indicating that exosomes play a critical role in intercellular communication and thus, have a great potential as biomarkers for a variety of diseases, including cancer, [5,6] immune disorders [7] and neurodegeneration. [8] MagCapture Exosome Isolation Kit PS can be used for isolating high purity exosomes as well as other extracellular vesicles from cell culture media or body fluids. This method enables rapid and antibody-free purification and does not require usage of an ultracentrifuge. PS AFFINITY METHOD A NOVEL PURIFICATION METHOD Conventional exosome isolation methods include ultracentrifugation (UC), polymer-based precipitation and affinity purification using surface antigen targeting antibodies. [4,9] Among those, UC although most commonly used requires the purchasing of an expensive instrument and allows purification of only small amounts of extracellular vesicles. [10] In contrast, polymer-based precipitation is rather inexpensive and can recover large amounts of exosomes. [11] Nonetheless, the purity of the isolated fractions is comparably low making this method unsuitable for most downstream applications. Affinity-based purification strategies use an antibody directed against exosome-specific surface proteins, such as CD9, CD63 or CD81. [12,13] Hence, these methods usually lead to a high purity of isolated fractions. One limitation, however, is the necessity of using stringent conditions to detach the antibodies from the purified exosomes. As a consequence, the isolated material is often damaged during the elution step, thus, making it unsuitable for downstream applications. MagCapture Exosome Isolation Kit PS has been recently introduced as an affinity-based, yet antibodyfree, purification method. It uses magnetic beads coupled to a phosphatidylserine (PS)-binding protein which recognizes and binds exosomes in a calcium-dependent manner. The elution of exosomes and extracellular vesicles can, therefore, be performed at neutral ph by simply adding a metal-chelating reagent. The intact exosomes isolated by means of this new purification method are suited for various applications including electron microscopy, nanoparticle tracking analysis and analysis of intra-vesicle proteins, lipids or nucleic acids. Metal ion Magnetic beads PS-binding protein Chelating agent Magnetic beads PS-binding protein Extracellular vesicle Intact extracellular vesicle Calcium-dependent capture of exosomes and extracellular vesicles 2

4 A NOVEL AFFINITY-BASED METHOD FOR EXOSOME ISOLATION COMPARISON WITH CONVENTIONAL METHODS The main advantage of the PS affinity method is the remarkably high purity and the high recovery of the isolated exosomes and extracellular vesicles. Conventional purification methods show lower performance results in terms of purity and material recovery. METHOD EXOSOME PURITY EXOSOME RECOVERY INTACT VESICLES RECOVERY PS affinity method Ultracentrifugation Polymer-based precipitation Exosome surface antigen affinity method (using antibodies) Very low Low Medium High Very High Yes Yes Yes No Comparison of the PS affinity method with conventional isolation methods The most commonly used method to purify extracellular vesicles is ultracentrifugation (UC). This purification method is time-consuming, cost-intensive and ineffective in terms of material recovery. Polymer-based precipitation, on the other hand, efficiently recovers exosomes, but is prone to contamination by non-exosomal particles and is, therefore, often not suitable for downstream applications. Another widely used method is the affinity purification using antibodies directed against exosomespecific surface proteins. Latter strategy, however, also suffers from poor compatibility with downstream applications, as harsh conditions are usually required to detach the purified vesicles from the antibody which often damage the isolated material. The newly developed PS affinity method is based on magnetic beads coated with a phosphatidylserine (PS)-binding receptor protein. Recognition of exosomes and extracellular vesicles occurs in a calciumdependent manner, thereby enabling easy elution of purified material using mild chelating agents. Hence, the PS affinity method allows an easy and high yielding purification of intact exosomes which can be further submitted to numerous downstream applications. MagCapture Exosome Isolation Kit PS Ultracentrifugation Polymer-based precipitation Electron microscopy pictures of extracellular vesicles purified by PS affinity method, UC and polymer-based precipitation 3

5 A NOVEL AFFINITY-BASED METHOD FOR EXOSOME ISOLATION MAGCAPTURE EXOSOME ISOLATION KIT PS PROTOCOL Sample pretreatment Cell culture medium Serum, plasma or urine Centrifugation Concentration (optional) Exosome binding enhancer (x500) Affinity reaction (180 min.) Incubation with functionalized magnetic beads Exosome elution buffer Washing buffer Exosome binding enhancer (x500) Washing & elution (15 min.) High purity and intact exosome Exosomes and extracellular vesicles isolation protocol using PS affinity method 4

6 A NOVEL AFFINITY-BASED METHOD FOR EXOSOME ISOLATION REFERENCES [1] M. Colombo, G. Raposo, C. Thery, Annu Rev Cell Dev Biol. 2014, 30, [2] M. Tkach, C. Thery, Cell. 2016, 164, [3] E. M. Kramer-Albers, A. F. Hill, Curr Opin Neurobiol. 2016, 39, [4] W. Nakai, T. Yoshida, D. Diez, Y. Miyatake, T. Nishibu, N. Imawaka, K. Naruse, Y. Sadamura, R. Hanayama, Sci Rep. 2016, 6, [5] J. Skog, T. Wurdinger, S. van Rijn, D. H. Meijer, L. Gainche, M. Sena-Esteves, W. T. Curry, Jr., B. S. Carter, A. M. Krichevsky, X. O. Breakefield, Nat Cell Biol. 2008, 10, [6] M. Baj-Krzyworzeka, R. Szatanek, K. Weglarczyk, J. Baran, B. Urbanowicz, P. Branski, M. Z. Ratajczak, M. Zembala, Cancer Immunol Immunother.: CII 2006, 55, [7] H. Valadi, K. Ekstrom, A. Bossios, M. Sjostrand, J. J. Lee, J. O. Lotvall, Nat Cell Biol. 2007, 9, [8] F. Raimondo, L. Morosi, C. Chinello, F. Magni, M. Pitto, Proteomics 2011, 11, [9] P. Li, M. Kaslan, S. H. Lee, J. Yao, Z. Gao, Theranostics 2017, 7, [10] C. Thery, S. Amigorena, G. Raposo, A. Clayton, Curr Protoc Cell Biol. 2006, Chapter 3, Unit [11] M. A. Rider, S. N. Hurwitz, D. G. Meckes, Sci Rep. 2016, 6, [12] L. Balaj, N. A. Atai, W. Chen, D. Mu, B. A. Tannous, X. O. Breakefield, J. Skog, C. A. Maguire, Sci Rep. 2015, 5, [13] A. Ghosh, M. Davey, I. C. Chute, S. G. Griffiths, S. Lewis, S. Chacko, D. Barnett, N. Crapoulet, S. Fournier, A. Joy, M. C. Caissie, A. D. Ferguson, M. Daigle, M. V. Meli, S. M. Lewis, R. J. Ouellette, PloS One 2014, 9, e PRODUCT DETAILS TTTTT PRODUCT NAME PACKAGE SIZE ARTICLE NO. STORAGE MagCapture Exosome Isolation Kit PS 2 Tests Tests Keep at 2-10 C 5

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