Ludwig, Anna-Kristin, De Miroschedji, Kyra, Doeppner, Thorsten R., Boerger, Verena, Ruesing, Johannes, Rebmann, Vera, Durst, Stephan, Jansen, Soeren, Bremer, Michel, Behrmann, Elmar ORCID: 0000-0001-6794-3669, Singer, Bernhard B., Jastrow, Holger, Kuhlmann, Jan Dominik, El Magraoui, Fouzi, Meyer, Helmut E., Hermann, Dirk M., Opalka, Bertram, Raunser, Stefan, Epple, Matthias ORCID: 0000-0002-1641-7068, Horn, Peter A. and Giebel, Bernd ORCID: 0000-0003-2446-948X (2018). Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales. J. Extracell. Vesicles, 7 (1). ABINGDON: TAYLOR & FRANCIS LTD. ISSN 2001-3078

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Abstract

Extracellular vesicles (EVs) provide a complex means of intercellular signalling between cells at local and distant sites, both within and between different organs. According to their cell-type specific signatures, EVs can function as a novel class of biomarkers for a variety of diseases, and can be used as drug-delivery vehicles. Furthermore, EVs from certain cell types exert beneficial effects in regenerative medicine and for immune modulation. Several techniques are available to harvest EVs from various body fluids or cell culture supernatants. Classically, differential centrifugation, density gradient centrifugation, size-exclusion chromatography and immunocapturing-based methods are used to harvest EVs from EV-containing liquids. Owing to limitations in the scalability of any of these methods, we designed and optimised a polyethylene glycol (PEG)based precipitation method to enrich EVs from cell culture supernatants. We demonstrate the reproducibility and scalability of this method and compared its efficacy with more classical EV-harvesting methods. We show that washing of the PEG pellet and the re-precipitation by ultracentrifugation remove a huge proportion of PEG co-precipitated molecules such as bovine serum albumine (BSA). However, supported by the results of the size exclusion chromatography, which revealed a higher purity in terms of particles per milligram protein of the obtained EV samples, PEG-prepared EV samples most likely still contain a certain percentage of other non-EV associated molecules. Since PEG-enriched EVs revealed the same therapeutic activity in an ischemic stroke model than corresponding cells, it is unlikely that such co-purified molecules negatively affect the functional properties of obtained EV samples. In summary, maybe not being the purification method of choice if molecular profiling of pure EV samples is intended, the optimised PEG protocol is a scalable and reproducible method, which can easily be adopted by laboratories equipped with an ultracentrifuge to enrich for functional active EVs.

Item Type: Journal Article
Creators:
CreatorsEmailORCIDORCID Put Code
Ludwig, Anna-KristinUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
De Miroschedji, KyraUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Doeppner, Thorsten R.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Boerger, VerenaUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Ruesing, JohannesUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Rebmann, VeraUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Durst, StephanUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Jansen, SoerenUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Bremer, MichelUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Behrmann, ElmarUNSPECIFIEDorcid.org/0000-0001-6794-3669UNSPECIFIED
Singer, Bernhard B.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Jastrow, HolgerUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Kuhlmann, Jan DominikUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
El Magraoui, FouziUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Meyer, Helmut E.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Hermann, Dirk M.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Opalka, BertramUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Raunser, StefanUNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Epple, MatthiasUNSPECIFIEDorcid.org/0000-0002-1641-7068UNSPECIFIED
Horn, Peter A.UNSPECIFIEDUNSPECIFIEDUNSPECIFIED
Giebel, BerndUNSPECIFIEDorcid.org/0000-0003-2446-948XUNSPECIFIED
URN: urn:nbn:de:hbz:38-169196
DOI: 10.1080/20013078.2018.1528109
Journal or Publication Title: J. Extracell. Vesicles
Volume: 7
Number: 1
Date: 2018
Publisher: TAYLOR & FRANCIS LTD
Place of Publication: ABINGDON
ISSN: 2001-3078
Language: English
Faculty: Unspecified
Divisions: Unspecified
Subjects: no entry
Uncontrolled Keywords:
KeywordsLanguage
CIRCULATING MICRORNAS; SAMPLE PREPARATION; EXOSOMES; INFLAMMATION; PROTEOME; DELIVERY; MARKERS; CELLS; SYSTEMS; FLUIDSMultiple languages
Cell BiologyMultiple languages
Refereed: Yes
URI: http://kups.ub.uni-koeln.de/id/eprint/16919

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