Elt University and by EFRO by way of the Interreg V Grensregio Vlaanderen Nederland project Trans Tech Diagnostics.LBP.Free flow electrophoresis allows preparation of EV fractions with high recovery and purity prices Gerhard Weber1, Robert Wildgruber1, Simon Staubach2, Robin Vaspin Proteins web Dittrich3, Peter Horn3, Verena Boerger4 and Bernd Giebel2 FFE Service; Carboxypeptidase A2 Proteins manufacturer 2Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany; Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden; 3Institute for Transfusion Medicine, University Hospital Essen, University of DuisburgEssen, Essen, German; 4Institute for Transfusion Medicine, University Hospital Essen, University Duisburg-Essen, Essen, GermanyIntroduction: Presently, it remains a challenge to prepare extracellular vesicles (EVs), specifically these from physique fluids, which include plasma, to high purity. Neither fractionation by density nor by size is adequate to separate EVs from all contaminants e.g. higher and low density lipoprotein (HDL/LDL) and also other contaminating elements. For now, a timeconsuming combination of two approaches (density and size) is expected to enrich EVs to high purities, on a regular basis resulting in low EV recoveries. Cost-free Flow Electrophoresis is actually a well-established preparative and micropreparative process to separate analytes with inherent difference of charge density and/or difference of pI-value. Methods: Totally free Flow Interval Zone Electrophoresis (FF-IZE), applying media of unique pH-values, ranging from pH = eight to pH = four.eight delivers most suitable protocols for the quantitative separation of amphoteric analytes,Thursday May 18,like proteins and peptides from non-amphoteric analytes like lipid vesicles, DNA and RNA. Results: Within our ongoing project we’ve optimized FF-IZE-pH protocols for the purification and isolation of EVs at the same time DNA and RNA from cell culture supernatants and human plasma samples. Upon screening for EV-specific samples within a dot blot method, EV-specific antigens are specifically recovered in a selected number of fractions. Currently, we characterize the identified fractions in a lot more detail. For the enumeration of ready EVs we make use of the Nanoparticle Tracking Analysis (NTA). Additionally, the presence of EV markers along with the absence of contaminants are analyzed by Western Blot. We document the appearance of isolated EVs by transmission electron microscopy and establish the miRNA profiles on the obtained fractions. Summary/Conclusion: The principle of FFE, the EV isolation strategy and our ongoing benefits will be presented.Funding Supported by the Polish National Centre for Investigation and Improvement STRATEGMED1/235773/19/NCBR/2016 “EXPLORE ME”.LBP.MicroRNA biogenesis and heterogeneous miRNA distribution in cancer EVs Nils J. Groenewegen, Catrin Lutz, Alba M. Losada, Monique A.J. van Eijndhoven and D. Michiel Pegtel Exosomes Study Group, Division of Pathology, VU University Medical Center, Amsterdam, The NetherlandsLBP.Visualization of extracellular vesicles derived from human bone marrow mesenchymal stem cells using fluorescent and magnetic labels; in vitro and in vivo studies Sylwia Koniusz1, Anna Andrzejewska1, Andrea Del Fattore2, Elbieta Karnas3, Malgorzata Frontczak-Baniewicz4, Hanna Kozlowska5, Maurizio Muraca6, Miroslaw Janowski7 and Barbara Lukomska1 NeuroRepair Department, Mossakowski Health-related Research Centre, PAS, Warsaw, Poland; 2Multifactorial Illness and Complicated Phenotype Study Location, Bambino GesChildren’.