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      Efficient ultrafiltration-based protocol to deplete extracellular vesicles from fetal bovine serum

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          ABSTRACT

          Fetal bovine serum (FBS) is the most commonly used supplement in studies involving cell-culture experiments. However, FBS contains large numbers of bovine extracellular vesicles (EVs), which hamper the analyses of secreted EVs from the cell type of preference and, thus, also the downstream analyses. Therefore, a prior elimination of EVs from FBS is crucial. However, the current methods of EV depletion by ultracentrifugation are cumbersome and the commercial alternatives expensive. In this study, our aim was to develop a protocol to completely deplete EVs from FBS, which may have wide applicability in cell-culture applications. We investigated different EV-depleted FBS prepared by our novel ultrafiltration-based protocol, by conventionally used overnight ultracentrifugation, or commercially available depleted FBS, and compared them with regular FBS. All sera were characterized by nanoparticle tracking analysis, electron microscopy, Western blotting and RNA quantification. Next, adipose-tissue mesenchymal stem cells (AT-MSCs) and cancer cells were grown in the media supplemented with the three different EV-depleted FBS and compared with cells grown in regular FBS media to assess the effects on cell proliferation, stress, differentiation and EV production. The novel ultrafiltration-based protocol depleted EVs from FBS clearly more efficiently than ultracentrifugation and commercial methods. Cell proliferation, stress, differentiation and EV production of AT-MSCs and cancer cell lines were similarly maintained in all three EV-depleted FBS media up to 96 h. In summary, our ultrafiltration protocol efficiently depletes EVs, is easy to use and maintains cell growth and metabolism. Since the method is also cost-effective and easy to standardize, it could be used in a wide range of cell-culture applications helping to increase comparability of EV research results between laboratories.

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          Most cited references14

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          Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication.

          Normal and malignant cells shed from their surface membranes as well as secrete from the endosomal membrane compartment circular membrane fragments called microvesicles (MV). MV that are released from viable cells are usually smaller in size compared to the apoptotic bodies derived from damaged cells and unlike them do not contain fragmented DNA. Growing experimental evidence indicates that MV are an underappreciated component of the cell environment and play an important pleiotropic role in many biological processes. Generally, MV are enriched in various bioactive molecules and may (i) directly stimulate cells as a kind of 'signaling complex', (ii) transfer membrane receptors, proteins, mRNA and organelles (e.g., mitochondria) between cells and finally (iii) deliver infectious agents into cells (e.g., human immuno deficiency virus, prions). In this review, we discuss the pleiotropic effects of MV that are important for communication between cells, as well as the role of MV in carcinogenesis, coagulation, immune responses and modulation of susceptibility/infectability of cells to retroviruses or prions.
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            Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes.

            Viable tumor cells actively release vesicles into the peripheral circulation and other biologic fluids, which exhibit proteins and RNAs characteristic of that cell. Our group demonstrated the presence of these extracellular vesicles of tumor origin within the peripheral circulation of cancer patients and proposed their utility for diagnosing the presence of tumors and monitoring their response to therapy in the 1970s. However, it has only been in the past 10 years that these vesicles have garnered interest based on the recognition that they serve as essential vehicles for intercellular communication, are key determinants of the immunosuppressive microenvironment observed in cancer and provide stability to tumor-derived components that can serve as diagnostic biomarkers. To date, the clinical utility of extracellular vesicles has been hampered by issues with nomenclature and methods of isolation. The term "exosomes" was introduced in 1981 to denote any nanometer-sized vesicles released outside the cell and to differentiate them from intracellular vesicles. Based on this original definition, we use "exosomes" as synonymous with "extracellular vesicles." While our original studies used ultracentrifugation to isolate these vesicles, we immediately became aware of the significant impact of the isolation method on the number, type, content and integrity of the vesicles isolated. In this review, we discuss and compare the most commonly utilized methods for purifying exosomes for post-isolation analyses. The exosomes derived from these approaches have been assessed for quantity and quality of specific RNA populations and specific marker proteins. These results suggest that, while each method purifies exosomal material, there are pros and cons of each and there are critical issues linked with centrifugation-based methods, including co-isolation of non-exosomal materials, damage to the vesicle's membrane structure and non-standardized parameters leading to qualitative and quantitative variability. The down-stream analyses of these resulting varying exosomes can yield misleading results and conclusions.
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              Stem Cell Extracellular Vesicles: Extended Messages of Regeneration

              Stem cells are critical to maintaining steady-state organ homeostasis and regenerating injured tissues. Recent intriguing reports implicate extracellular vesicles (EVs) as carriers for the distribution of morphogens and growth and differentiation factors from tissue parenchymal cells to stem cells, and conversely, stem cell–derived EVs carrying certain proteins and nucleic acids can support healing of injured tissues. We describe approaches to make use of engineered EVs as technology platforms in therapeutics and diagnostics in the context of stem cells. For some regenerative therapies, natural and engineered EVs from stem cells may be superior to single-molecule drugs, biologics, whole cells, and synthetic liposome or nanoparticle formulations because of the ease of bioengineering with multiple factors while retaining superior biocompatibility and biostability and posing fewer risks for abnormal differentiation or neoplastic transformation. Finally, we provide an overview of current challenges and future directions of EVs as potential therapeutic alternatives to cells for clinical applications.
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                Author and article information

                Journal
                J Extracell Vesicles
                J Extracell Vesicles
                ZJEV
                zjev20
                Journal of Extracellular Vesicles
                Taylor & Francis
                2001-3078
                2018
                21 January 2018
                : 7
                : 1
                : 1422674
                Affiliations
                [ a ] Department of Oral and Maxillofacial Diseases, University of Helsinki and Helsinki University Hospital , Helsinki, Finland
                [ b ] EV core and Institute for Molecular Medicine, Finland FIMM, University of Helsinki , Helsinki, Finland
                [ c ] Laser Tilkka Ltd , Helsinki, Finland
                [ d ] EV Core and Division of Biochemistry and Biotechnology and Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki , Helsinki, Finland
                Author notes
                CONTACT Sippy Kaur sippy.kaur@ 123456helsinki.fi Department of Oral and Maxillofacial Diseases, University of Helsinki and Helsinki University Hospital , PO Box 63, 00014 University of Helsinki, Finland
                [*]

                These authors are shared authors.

                [†]

                These authors contributed equally to this work

                Author information
                http://orcid.org/0000-0003-2326-5821
                Article
                1422674
                10.1080/20013078.2017.1422674
                5795649
                29410778
                5b441f01-3111-4912-8cbd-a7117133f43e
                © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License ( http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 16 March 2017
                : 23 December 2017
                Page count
                Figures: 8, Tables: 3, References: 34, Pages: 14
                Funding
                Funded by: Biotieteiden ja Ympäristön Tutkimuksen Toimikunta 10.13039/501100005876
                Award ID: 287089
                Funded by: Helsingin ja Uudenmaan Sairaanhoitopiiri 10.13039/100008376
                Award ID: TYH2016130
                Funded by: Helsingin ja Uudenmaan Sairaanhoitopiiri 10.13039/100008376
                Award ID: Y1014SUL05
                Funded by: Helsingin ja Uudenmaan Sairaanhoitopiiri 10.13039/100008376
                Award ID: Y1014SUL05
                Funded by: Helsingin ja Uudenmaan Sairaanhoitopiiri 10.13039/100008376
                Award ID: TYH2016130
                Funded by: Private donation/University of Helsinki
                Award ID: WBS73714301
                Funded by: Tekes 10.13039/501100003406
                Award ID: 5773/31/16
                Funded by: Tekes 10.13039/501100003406
                Award ID: 3986/31/2013
                Funded by: Tekes 10.13039/501100003406
                Award ID: 40294/11
                Funded by: Tekes 10.13039/501100003406
                Award ID: 5773/31/16
                This work was supported by the Biotieteiden ja Ympäristön Tutkimuksen Toimikunta [287089]; Helsingin ja Uudenmaan Sairaanhoitopiiri [Y1014SUL05]; Helsingin ja Uudenmaan Sairaanhoitopiiri [TYH2016130]; Helsingin ja Uudenmaan Sairaanhoitopiiri [Y1014SUL05]; Helsingin ja Uudenmaan Sairaanhoitopiiri [TYH2016130]; Selma and Maja-Lisa Selander Foundation; Evald and Hilda Nissi Foundation; private donation/University of Helsinki [WBS73714301]; Paulon Säätiö; Suomen Hammaslääkäriseura Apollonia; Tekes [5773/31/16]; Tekes [3986/31/2013]; Tekes [40294/11]; Tekes [5773/31/16].
                Categories
                Research Article
                Research Article

                adipose-tissue mesenchymal stem cells,extracellular vesicles,fetal bovine serum,depleted serum,ultracentrifugation,ultrafiltration

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