UNCORRECTED PROOF 1 Ultrafiltration with size-exclusion liquid chromatography for high yield 2 isolation of extracellular vesicles preserving intact biophysical and 3 functional properties 4 Q1 Joel Z. Nordin, M.D. a,1 , Yi Lee, BSc b,1 , Pieter Vader, PhD b , Imre Mäger, PhD b,c , 5 Henrik J. Johansson, PhD d , Wolf Heusermann, PhD e , Oscar P.B. Wiklander, M.D. a Q2 Q3 , 6 Mattias Hällbrink, PhD f , Yiqi Seow, DPhil g , Jarred J. Bultema, PhD h , Jonathan Gilthorpe, PhD i , 7 Tim Davies, MSc j , Paul J. Fairchild, PhD j , Susanne Gabrielsson, PhD h , 8 Nicole C. Meisner-Kober, PhD e , Janne Lehtiö, PhD d , C.I. Edvard Smith, M.D., PhD a , 9 Matthew J.A. Wood, M.D., DPhil b, ⁎ , Samir E.L. Andaloussi, PhD a,b,⁎⁎ 10 a Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden 11 b Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom 12 c Institute of Technology, University of Tartu, Tartu, Estonia 13 d Cancer Proteomics Mass Spectrometry, Science for Life Laboratory, Department of Oncology-Pathology, Karolinska Institutet, Stockholm, Sweden 14 e Novartis Institutes for Biomedical Research, Basel, Switzerland 15 f Department of Neurochemistry, Stockholm University, Stockholm, Sweden 16 g Molecular Engineering Laboratory, Proteos, Agency for Science, Technology and Research (A*STAR), Singapore 17 h Department of Medicine Solna, Translational Immunology Unit, Karolinska Institutet and University Hospital, Stockholm, Sweden 18 i Umeå Centre for Molecular Medicine, Umeå University, Umeå, Sweden 19 j Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom 20 Received 19 November 2014; accepted 4 January 2015 21 Abstract 22 Extracellular vesicles (EVs) are natural nanoparticles that mediate intercellular transfer of RNA and proteins and are of great medical 23 interest; serving as novel biomarkers and potential therapeutic agents. However, there is little consensus on the most appropriate method to 24 isolate high-yield and high-purity EVs from various biological fluids. Here, we describe a systematic comparison between two protocols for 25 EV purification: ultrafiltration with subsequent liquid chromatography (UF-LC) and differential ultracentrifugation (UC). A significantly 26 higher EV yield resulted from UF-LC as compared to UC, without affecting vesicle protein composition. Importantly, we provide novel 27 evidence that, in contrast to UC-purified EVs, the biophysical properties of UF-LC-purified EVs are preserved, leading to a different in vivo 28 biodistribution, with less accumulation in lungs. Finally, we show that UF-LC is scalable and adaptable for EV isolation from complex media 29 types such as stem cell media, which is of huge significance for future clinical applications involving EVs. Q4 30 © 2015 Published by Elsevier Inc. 31 Key words: Biophysical properties; extracellular vesicles; size-exclusion liquid chromatography; ultracentrifugation; ultrafiltration 32 Nanomedicine: Nanotechnology, Biology, and Medicine xx (2015) xxx – xxx nanomedjournal.com Statements of funding: The project was funded by the Swedish Research Council (VR-Med) and Swedish Cancer society. SEA holds a research fellowship from the Swedish Society of Medical Research (SSMF). SG holds a position from the Swedish Heart-Lung Foundation. JN and OW are both recipients of Karolinska Institutet MD/PhD grants. YL is funded by the Agency for Science, Technology and Research (A*STAR), Singapore. IM is supported by a Postdoctoral MOBILITAS Fellowship of the Estonian Science Foundation and by the EU IMI (Innovative Medicines Initiative) project COMPACT (Collaboration on the optimisation of macromolecular pharmaceutical access to cellular targets). Competing financial interests: The authors declare no competing financial interests. ⁎ Correspondence to: M. JA Wood, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom. ⁎⁎ Correspondence to: S. El Andaloussi, Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Sweden. E-mail addresses: matthew.wood@dpag.ox.ac.uk (M.J.A. Wood), Samir.El-Andaloussi@ki.se (S.E.L. Andaloussi). 1 These authors contributed equally to the manuscript. http://dx.doi.org/10.1016/j.nano.2015.01.003 1549-9634/© 2015 Published by Elsevier Inc. NANO-01055; No of Pages 5 Please cite this article as: Nordin J.Z., et al., Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties. Nanomedicine: NBM 2015;xx:1-5, http://dx.doi.org/10.1016/j.nano.2015.01.003