The NanoMatFutur Junior Research program from the Federal Ministry of Education and Research, Germany (grant number 13XP5029A) supported this work. Maximilian Richter was supported by Studienstiftung des Deutschen Volkes (German Academic Scholarship Foundation) through a Ph.D. fellowship.

1. Cell culture and the production of cell-conditioned medium
<ol>
	<li>Generally, cultivate cells under the individual conditions required for the respective cell line.</li>
	<li>Cultivate the cells for 24-72 h in serum-free conditions or in medium containing EV-depleted fetal bovine serum (FBS). 
	NOTE: If EV-depleted FBS is used, employ a method proven to efficiently deplete the serum, to prevent contamination with bovine serum-derived EVs26.</li>
	<li>Collect the medium from the flasks....

<ol>
	<li>Stremersch, S., De Smedt, S. C., Raemdonck, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+and+diagnostic+applications+of+extracellular+vesicles.">Therapeutic and diagnostic applications of extracellular vesicles.</a> Journal of Controlled Release. 244, 167-183 (2016).</li><li>Fuhrmann, G., Herrmann, I. K., Stevens, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-derived+vesicles+for+drug+therapy+and+diagnostics:+Opportunities+and+challenges.">Cell-derived vesicles for drug therapy and diagnostics: Opportunities and challenges.</a> Nano Today. 10 (3), 397-409 (2015).</li><li>Goes, A., Fuhrmann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenic+and+Biomimetic+Carriers+as+Versatile+Transporters+To+Treat+Infections.">Biogenic and Biomimetic Carriers as Versatile Transporters To Treat Infections.</a> ACS Infectious Diseases. 4 (6), 881-892 (2018).</li><li>Gy&#246;rgy, B., Hung, M. E., Breakefield, X. O., Leonard, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+Applications+of+Extracellular+Vesicles:+Clinical+Promise+and+Open+Questions.">Therapeutic Applications of Extracellular Vesicles: Clinical Promise and Open Questions.</a> Annual Review of Pharmacology and Toxicology. 55, 439-464 (2015).</li><li>Schulz, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocompatible+bacteria-derived+vesicles+show+inherent+antimicrobial+activity.">Biocompatible bacteria-derived vesicles show inherent antimicrobial activity.</a> Journal of Controlled Release. 290, 46-55 (2018).</li><li>Feng, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+class+of+extracellular+vesicles+from+breast+cancer+cells+activates+VEGF+receptors+and+tumour+angiogenesis.">A class of extracellular vesicles from breast cancer cells activates VEGF receptors and tumour angiogenesis.</a> Nature Communications. 8, 14450 (2017).</li><li>Bewicke-Copley, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+released+following+heat+stress+induce+bystander+effect+in+unstressed+populations.">Extracellular vesicles released following heat stress induce bystander effect in unstressed populations.</a> Journal of Extracellular Vesicles. 6, 1340746 (2017).</li><li>Xu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+transfer+antigens+to+dendritic+cells+by+releasing+exosomes+containing+dead-cell-associated+antigens+partially+through+a+ceramide-dependent+pathway+to+enhance+CD4(+)+T-cell+responses.">Macrophages transfer antigens to dendritic cells by releasing exosomes containing dead-cell-associated antigens partially through a ceramide-dependent pathway to enhance CD4(+) T-cell responses.</a> Immunology. 149 (2), 157-171 (2016).</li><li>Buzas, E. I., Gy&#246;rgy, B., Nagy, G., Falus, A., Gay, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+role+of+extracellular+vesicles+in+inflammatory+diseases.">Emerging role of extracellular vesicles in inflammatory diseases.</a> Nature Reviews Rheumatology. 10, 356 (2014).</li><li>Rajappa, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+Astrocytic+Tumor+Progression+Potentiated+by+JAK-mediated+Recruitment+of+Myeloid+Cells.">Malignant Astrocytic Tumor Progression Potentiated by JAK-mediated Recruitment of Myeloid Cells.</a> Clinical Cancer Research: An Official Journal of the American Association for Cancer Research. 23 (12), 3109-3119 (2017).</li><li>Umezu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomal+miR-135b+shed+from+hypoxic+multiple+myeloma+cells+enhances+angiogenesis+by+targeting+factor-inhibiting+HIF-1.">Exosomal miR-135b shed from hypoxic multiple myeloma cells enhances angiogenesis by targeting factor-inhibiting HIF-1.</a> Blood. 124 (25), 3748-3757 (2014).</li><li>Costa-Silva, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+cancer+exosomes+initiate+pre-metastatic+niche+formation+in+the+liver.">Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver.</a> Nature Cell Biology. 17 (6), 816-826 (2015).</li><li>Boulanger, C. M., Loyer, X., Rautou, P. -. E., Amabile, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+in+coronary+artery+disease.">Extracellular vesicles in coronary artery disease.</a> Nature Reviews Cardiology. 14, 259 (2017).</li><li>Zhu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+toxicity+and+immunogenicity+studies+reveal+minimal+effects+in+mice+following+sustained+dosing+of+extracellular+vesicles+derived+from+HEK293T+cells.">Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from HEK293T cells.</a> Journal of Extracellular Vesicles. 6 (1), 1324730 (2017).</li><li>Vader, P., Mol, E. A., Pasterkamp, G., Schiffelers, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+for+drug+delivery.">Extracellular vesicles for drug delivery.</a> Advanced Drug Delivery Reviews. 106, 148-156 (2016).</li><li>Ingato, D., Lee, J. U., Sim, S. J., Kwon, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Good+things+come+in+small+packages:+Overcoming+challenges+to+harness+extracellular+vesicles+for+therapeutic+delivery.">Good things come in small packages: Overcoming challenges to harness extracellular vesicles for therapeutic delivery.</a> Journal of Controlled Release. 241, 174-185 (2016).</li><li>Gimona, M., Pachler, K., Laner-Plamberger, S., Schallmoser, K., Rohde, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manufacturing+of+Human+Extracellular+Vesicle-Based+Therapeutics+for+Clinical+Use.">Manufacturing of Human Extracellular Vesicle-Based Therapeutics for Clinical Use.</a> International Journal of Molecular Sciences. 18 (6), 1190 (2017).</li><li>Jeyaram, A., Jay, S. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+storage+on+physical+and+functional+properties+of+extracellular+vesicles+derived+from+neutrophilic+granulocytes.">Effect of storage on physical and functional properties of extracellular vesicles derived from neutrophilic granulocytes.</a> Journal of Extracellular Vesicles. 3, 25465 (2014).</li><li>Kreke, M., Smith, R., Hanscome, P., Peck, K., Ibrahim, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processes+for+producing+stable+exosome+formulations.">Processes for producing stable exosome formulations.</a> US patent. , (2016).</li><li>Frank, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles+protect+glucuronidase+model+enzymes+during+freeze-drying.">Extracellular vesicles protect glucuronidase model enzymes during freeze-drying.</a> Scientific Reports. 8 (1), 12377 (2018).</li><li>Charoenviriyakul, C., Takahashi, Y., Nishikawa, M., Takakura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+exosomes+at+room+temperature+using+lyophilization.">Preservation of exosomes at room temperature using lyophilization.</a> International Journal of Pharmaceutics. 553 (1), 1-7 (2018).</li><li>Kusuma, G. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+Protect+and+to+Preserve:+Novel+Preservation+Strategies+for+Extracellular+Vesicles.">To Protect and to Preserve: Novel Preservation Strategies for Extracellular Vesicles.</a> Frontiers in Pharmacology. 9 (1199), (2018).</li><li>Haney, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+as+drug+delivery+vehicles+for+Parkinson's+disease+therapy.">Exosomes as drug delivery vehicles for Parkinson's disease therapy.</a> Journal of Controlled Release. 207, 18-30 (2015).</li><li>Fuhrmann, G., Serio, A., Mazo, M., Nair, R., Stevens, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+loading+into+extracellular+vesicles+significantly+improves+the+cellular+uptake+and+photodynamic+effect+of+porphyrins.">Active loading into extracellular vesicles significantly improves the cellular uptake and photodynamic effect of porphyrins.</a> Journal of Controlled Release. 205, 35-44 (2015).</li><li>Th&#233;ry, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+information+for+studies+of+extracellular+vesicles+2018+(MISEV2018):+a+position+statement+of+the+International+Society+for+Extracellular+Vesicles+and+update+of+the+MISEV2014+guidelines.">Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.</a> Journal of Extracellular Vesicles. 7 (1), 1535750 (2018).</li><li>Gardiner, C., Ferreira, Y. J., Dragovic, R. A., Redman, C. W. G., Sargent, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicle+sizing+and+enumeration+by+nanoparticle+tracking+analysis.">Extracellular vesicle sizing and enumeration by nanoparticle tracking analysis.</a> Journal of Extracellular Vesicles. 2 (1), 19671 (2013).</li><li>Vestad, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+and+concentration+analyses+of+extracellular+vesicles+by+nanoparticle+tracking+analysis:+a+variation+study.">Size and concentration analyses of extracellular vesicles by nanoparticle tracking analysis: a variation study.</a> Journal of Extracellular Vesicles. 6 (1), 1344087 (2017).</li><li>Bosch, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trehalose+prevents+aggregation+of+exosomes+and+cryodamage.">Trehalose prevents aggregation of exosomes and cryodamage.</a> Scientific Reports. 6, 36162 (2016).</li><li>Bhattacharjee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DLS+and+zeta+potential+-+What+they+are+and+what+they+are+not.">DLS and zeta potential - What they are and what they are not.</a> Journal of Controlled Release. 235, 337-351 (2016).</li><li>Van Deun, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+disparate+isolation+methods+for+extracellular+vesicles+on+downstream+RNA+profiling.">The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling.</a> Journal of Extracellular Vesicles. 3 (1), 24858 (2014).</li><li>Taylor, D. 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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+cell+culture+parameters+on+production+and+vascularization+bioactivity+of+mesenchymal+stem+cell-derived+extracellular+vesicles.">Impact of cell culture parameters on production and vascularization bioactivity of mesenchymal stem cell-derived extracellular vesicles.</a> Bioengineering &amp; Translational Medicine. 2 (2), 170-179 (2017).</li><li>Gardiner, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+used+for+the+isolation+and+characterization+of+extracellular+vesicles:+results+of+a+worldwide+survey.">Techniques used for the isolation and characterization of extracellular vesicles: results of a worldwide survey.</a> Journal of Extracellular Vesicles. 5 (1), 32945 (2016).</li><li>Zhang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+distinct+nanoparticles+and+subsets+of+extracellular+vesicles+by+asymmetric+flow+field-flow+fractionation.">Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation.</a> Nature Cell Biology. 20 (3), 332-343 (2018).</li><li>Linares, R., Tan, S., Gounou, C., Arraud, N., Brisson, A. 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The authors have nothing to disclose.

In this manuscript, we present a comprehensive protocol to study the stability of EVs under different storage conditions. With the combination of encapsulated glucuronidase as a functional readout and the evaluation of the physicochemical parameters of the EVs, the protocol allows for a straightforward storage stability evaluation of EVs and the comparison of EVs from different cell lines. SEM and TEM as complementary methods allow an insight into changes of the EVs on the single-particle level. The results presented her...

EVs are membrane-bound nanoparticles produced by nearly all cell types. For mammalian cells, EVs can be subdivided into two main groups with distinct production pathways1,2. Membrane vesicles, with a size range from roughly 100-1,000 nm, are produced by direct budding from the cell membrane. Exosomes, sized 30-200 nm, are derived from multivesicular bodies formed by inward budding into endosomes that subsequently fuse with the cell membrane to release multiple exosomes at once. The main function of these vesicles is the transport of information between cells3. For this purpose, cargos such as RNA, DNA, and proteins are actively sorted into them. EVs can convey a variety of effects on their targets, with implications for both health and disease state. On one side, they mediate positive effects such as tissue regeneration, antigen presentation, or antibiotic effects, which makes them auspicious targets for their development as therapeutics4,5. On the other side, EVs can promote tumor vascularization6, induce bystander effects in stress responses7, and might play a role in autoimmune diseases8 and inflammatory diseases9. Thus, they might be a key component to a better understanding of many pathological effects. However, the presence of altered EVs in manifold diseases, such as cancer10,11,12 and cardiovascular disorders13, and their easy accessibility in blood and urine makes them ideal biomarkers. Finally, their good biocompatibility14 and their inherent targeting ability make EVs also interesting for drug delivery15. In this manuscript, we describe a protocol for the evaluation of the storage stability of EVs derived from mammalian cells, an important property that is still little investigated.
For the clinical development of EVs, there are still many obstacles to surmount16, including the evaluation of their therapeutic effects, production, purification, and storage17. While -80 &#176;C is widely seen as the gold standard for EV storage18, the required freezers are expensive, and maintaining the required cold chain from the production to the patient can be challenging. Moreover, some reports indicate that storage at -80 &#176;C still not optimally preserves EVs and induces a loss in EV functionality19,20. Other methods, such as freeze-drying21,22 or spray-drying23, have been proposed as potential alternatives to the frozen storage of EVs.
The optimal way of assessing storage stability would be to test the EVs in functional assays or by the evaluation of a specific marker, for instance, their antibacterial activity19. This is possible when the desired effect of the vesicles is known and when one distinct group of EVs is to be studied. If EVs from different cell sources are to be compared (e.g., for drug encapsulation) or if there is no known functional readout, it is no longer possible to assess changes due to storage in a direct manner.
On the other hand, simply evaluating changes in their physicochemical parameters, such as size, particle recovery, and protein concentration, does not always predict changes in EV activity, as has been shown in a recent patent20.
Here, we provide a readily applicable protocol for measuring the storage stability of EVs by assessing their physicochemical parameters combined with the activity of an encapsulated beta-glucuronidase enzyme as a surrogate for the cargo of the EVs. The loading of the enzyme is done by saponin incubation, a mild method established with EVs from different sources21,24,25. Saponin forms transient pores in the EV membrane, which allows enzyme uptake into the vesicle. As enzymes are prone to lose their activity if subjected to unfavorable storage conditions, they are an ideal surrogate for the evaluation of the preservation of functional cargoes of the EVs.
We have demonstrated that the application of this protocol to EVs derived from human mesenchymal stem cells (MSCs), human umbilical vein endothelial cells (HUVECs), and human adenocarcinoma alveolar epithelial cells (A549) indeed result in great differences in storage stability between different cell lines, which should be taken into consideration when choosing the EV source21.

<table border="0" cellpadding="0" cellspacing="0" style="width:933px;" width="933">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:333px;">1,2 dimyristoyl-sn glycero-3-phospho-choline (DMPC)</td>
			<td style="width:315px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2663-25MG</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:333px;">1,2-dipalmitoyl-sn-glycero-3-phospho-choline (DPPC)</td>
			<td style="width:315px;">Sigma-Aldrich</td>
			<td>P4329-25MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">225 cm&#178; cell culture flasks</td>
			<td style="width:315px;">Corning</td>
			<td>431082</td>
			<td>Used with 25 ml of medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">30 kDa regenerated cellulose membrane</td>
			<td style="width:315px;">Wyatt Technology Europe</td>
			<td>1854</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">350 &#181;m spacer</td>
			<td style="width:315px;">Wyatt Technology Europe</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Automated fraction collector</td>
			<td style="width:315px;">Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Beta-glucuronidase</td>
			<td style="width:315px;">Sigma-Aldrich</td>
			<td style="width:119px;">G7646-100KU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Chloroform</td>
			<td style="width:315px;">Fisher scientific</td>
			<td>C/4966/17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Column oven</td>
			<td style="width:315px;">Hitachi High-Technologies Europe</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">D-(+)-Trehalose dihydrate</td>
			<td style="width:315px;">Sigma-Aldrich</td>
			<td>T9531-10G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:333px;">DAWN HELEOS II, Multi-angle light scattering detector</td>
			<td style="width:315px;">&#160;Wyatt Technology Europe</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Durapore Membrane filter, PVDF,&#160; 0,1&#160;&#181;m, 47&#160;mm</td>
			<td style="width:315px;">Merck</td>
			<td>VVLP04700</td>
			<td>Used for the preparation of buffers for AF4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">EBM-2</td>
			<td style="width:315px;">Lonza Verviers, S.p.r.</td>
			<td>CC-3156</td>
			<td>Endothelial Cell Growth basal medium, used for the serum free culture of HUVEC cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Eclipse dualtec</td>
			<td style="width:315px;">Wyatt Technology Europe</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">EGM-2</td>
			<td style="width:315px;">Lonza Verviers, S.p.r.</td>
			<td>CC-3162</td>
			<td>Endothelial Cell Growth medium, used for the normal culture of HUVEC cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ELISA Plate Sealers</td>
			<td>R&#38;D Systems</td>
			<td>DY992</td>
			<td>used for sealing of 96-well plates for the glucuronidase assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Ethanol</td>
			<td style="width:315px;">Fisher scientific</td>
			<td>E/0665DF/17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Extruder Set With Holder/Heating Block</td>
			<td style="width:315px;">Avanti Polar Lipids</td>
			<td>610000-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Filter support</td>
			<td style="width:315px;">Avanti Polar Lipids</td>
			<td>610014-1EA</td>
			<td>used for liposome preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Fluorescein di-&#946;-D-glucoronide</td>
			<td style="width:315px;">Thermo Fisher Scientific</td>
			<td>F2915</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Gibco PBS-tablets+CA10:F36</td>
			<td style="width:315px;">Thermo Fisher Scientific</td>
			<td>18912014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Hettich Universal 320 R</td>
			<td>Andreas Hettich GmbH &#38; Co.KG</td>
			<td style="width:119px;"></td>
			<td>Used for pelleting cells at 300 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Hettich Rotina 420 R</td>
			<td style="width:315px;">Andreas Hettich GmbH &#38; Co.KG</td>
			<td style="width:119px;"></td>
			<td>Used for pelleting larger debris at 3000 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">HUVEC cells</td>
			<td style="width:315px;">Lonza Verviers, S.p.r.</td>
			<td style="width:119px;">C2517A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Kimble&#160; FlexColumn 1X30CM</td>
			<td style="width:315px;">Kimble</td>
			<td>420401-1030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Lyophilizer ALPHA 2-4 LSC</td>
			<td style="width:315px;">Christ</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge Tubes, Polypropylene</td>
			<td>VWR international</td>
			<td style="width:119px;">525-0255</td>
			<td>the tubes used for all EV-handling, found to be more favorable than comparable products from other suppliers regarding particle recovery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Nanosight LM14 equipped with a green laser</td>
			<td style="width:315px;">Malvern Pananalytical</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Nanosight-software version 3.1</td>
			<td style="width:315px;">Malvern Pananalytical</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:333px;">Nucleopore 200 nm track-etch polycarbonate membranes</td>
			<td>Whatman/GE Healthcare</td>
			<td align="right">110406</td>
			<td>used for liposome preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">PEEK Inline filter holder</td>
			<td style="width:315px;">Wyatt Technology Europe</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphotungstic acid hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>79690-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Polycarbonate bottles for ultracentrifugation</td>
			<td style="width:315px;">Beckman Coulter</td>
			<td style="width:119px;">355622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuantiPro BCA Assay Kit</td>
			<td style="width:315px;">Sigma-Aldrich</td>
			<td>QPBCA-1KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Saponin</td>
			<td style="width:315px;">Sigma-Aldrich</td>
			<td>47036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Scanning electron microscopy Zeiss EVO HD 15</td>
			<td style="width:315px;">Carl Zeiss AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Sepharose Cl-2b</td>
			<td style="width:315px;">GE Healthcare</td>
			<td>17014001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">SEM copper grids with carbon film</td>
			<td style="width:315px;">Plano</td>
			<td>S160-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Small AF4 channel</td>
			<td style="width:315px;">Wyatt Technology Europe</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Sputter-coater Q150R ES</td>
			<td style="width:315px;">Quorum Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:333px;">Transmission electron microscopy JEOL JEM 2011</td>
			<td style="width:315px;">Oxford Instruments</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Type 45 Ti ultracentrifugation rotor</td>
			<td style="width:315px;">Beckman Coulter</td>
			<td>339160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Ultimate 3000 Dionex autosampler</td>
			<td style="width:315px;">Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Ultimate 3000 Dionex isocratic pump</td>
			<td style="width:315px;">Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Ultimate 3000 Dionex online vacuum degasser</td>
			<td style="width:315px;">Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Ultracentrifuge OptimaTM L-90 K</td>
			<td style="width:315px;">Beckman Coulter</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">UV detector</td>
			<td style="width:315px;">Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:333px;">Whatman 0.2 &#181;m pore size mixed cellulose filter</td>
			<td>Whatman/GE Healthcare</td>
			<td style="width:119px;">10401712</td>
			<td>Used for the filtration of all buffers used with the EVs and in SEC</td>
		</tr>
	</tbody>
</table>

evaluation of the storage stability of extracellular vesicles

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical development, not only their pharmaceutical activity is important but also their production needs to be evaluated. In this context, research focuses on the isolation of EVs, their characterization, and their storage. The present manuscript aims at providing a facile procedure for the assessment of the effect of different storage conditions on EVs, without genetic manipulation or specific functional assays. This makes it possible to quickly get a first impression of the stability of EVs under a given storage condition, and EVs derived from different cell sources can be compared easily. The stability measurement is based on the physicochemical parameters of the EVs (size, particle concentration, and morphology) and the preservation of the activity of their cargo. The latter is assessed by the saponin-mediated encapsulation of the enzyme beta-glucuronidase into the EVs. Glucuronidase acts as a surrogate and allows for an easy quantification via the cleavage of a fluorescent reporter molecule. The present protocol could be a tool for researchers in the search for storage conditions that optimally retain EV properties to advance EV research toward clinical application.

Figure 1 displays the storage characteristics of EVs isolated from HUVECs. EVs were isolated by UC, glucuronidase was encapsulated, and after SEC, the purified EVs were evaluated for their physicochemical properties by NTA. A sample of the vesicles was subsequently subjected to AF4 purification and the glucuronidase activity was measured.
The vesicles were then stored for 7 d at 4 &#176;C or -80 &#176;C and at 4 &#176;C in lyophilized form, in the latter case with the addition...

Watch this Scientific Journal Video about Evaluation of the Storage Stability of Extracellular Vesicles at JoVE.com

Evaluation of the Storage Stability of Extracellular Vesicles

Here we present a readily applicable protocol to assess the storage stability of extracellular vesicles, a group of naturally occurring nanoparticles produced by cells. The vesicles are loaded with glucuronidase as a model enzyme and stored under different conditions. After storage, their physicochemical parameters and the activity of the encapsulated enzyme are evaluated.

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical ...

The storage stability of extra cellular vesicles is an important parameter for their prospective therapeutic use. Our protocol provides a readily applicable tool for evaluating how storage effects the vesicles. The main advantage of our technique is that by introducing glucuronidase as an exogenous marker, extracellular vesicles derived from different parent cells an be easily compared regarding their storability.Asymmetric flow field flow fractionation, or AF4, is a mild alternative to size exclusion chromatography purification for very sensitive compounds. Because the compounds encounter less sheer stress in the channel. As glucuronidase is a sensitive enzyme, the quality of the resides benefits from thorough planning and carrying out the steps for purification and analysis back to back.After culturing the cell line of interest according to standard conditions for those cells, replace the supernatant with serum free or extra cellular vesicle depleted medium and return the cells to the cell culture incubator for an additional 24 to 72 hours. At the end of the incubation, collect the super natant from each flask and centrifuge the samples to sediment the cells. Carefully collect the cell conditioned medium without disturbing the pellet.And centrifuge the medium again to remove cell debris and large aggregates. Then carefully transfer the supernatants into ultracentrifuge tubes. If using a fixed angle rotor, mark the orientation of the tubes in the centrifuge to facilitate the retrieval of the extracellular vesicle pellet after the centrifugation.At the end of the ultracentrifugation, use a serological pipette to carefully discard the supernatant without disturbing the extracellular vesicle pellet. At 200 microliters of 0.2 micrometer pore filtered PBS to the residual supernatant of the first ultracentrifuge tube. After resuspending the pellet, transfer the resulting extracellular vesicle suspension to the next tube for resuspension of the subsequent pellet until all of the extracellular vesicle pellets have been resuspended.Add beta glucuronidase to the final resuspended pellet solution to a final concentration of 1.5 milligrams per milliliter. And saponin to a final concentration of 0.1 milligrams per milliliter. Then mix well by vortexing for three seconds and place the reaction at room temperature for 10 minutes with intermittent gentle flicking.Once the glucuronidase has been encapsulated, be sure to perform all of the subsequent purification vesicle evaluation steps on the same day to obtain unbiased storage stability results. Have a size exclusion chromatography column equilibrated with at least two column volumes of PBS ready to purify the pellet immediately after the saponin incubation. To purify, add up to 400 microliters of extracellular vesicle suspension to the column.Then collect one milliliter fractions of the eluate. To conform the separation of the extracellular vesicles from the contaminating proteins and free glucuronidase, measure the protein concentration by bicinchoninic acid assay according to the manufacturers instructions. Using parameters optimized for the respective instrument and vesicle type, use a nano particle tracking analysis instrument to measure the extracellular vesicle size and concentration.To measure the glucuronidase activity, add 25 microliters of freshly prepared fluorescin di-beta-D-glucuronide to 125 microliters of the purified extracellular vesicles and add the sample to one well of a black 96-well plate. Measure the time zero fluorescin production on a plate reader at a 480 nanometer excitation and 516 nanometer emission. Cover the plate tightly with transparent plastic foil to reduce evaporation.Then incubate the plate protected from light for 18 hours at 37 degrees Celsius. Measure the 18 hour fluorescin production on the plate reader as just demonstrated. For extracellular vesicle lyophilization, add four milligrams per milliliter of trehalose to the purified vesicles.And freeze the vesicles at minus 80 degrees Celsius for at least one hour. To lyophilize the samples, set the shelf temperature of a lyophilizer to 15 degrees Celsius and the pressure to 0.180 millibars. Dry the vesicles under these conditions for 46 hours.At the end of the main drying step, set the shelf temperature to 25 degrees Celsius and the pressure to 0035 millibars and dry the samples for two hours under these conditions. Then store the lyophilized samples at four degrees Celsius. To rehydrate the samples after storage, add a volume of water equal to the volume of the extracellular vesicle suspension prior to the lyophilization.To assess the enzyme activity after storage, load an AF4 instrument with freshly prepared 0.1 micrometer filtered PBS for the mobile phase and insert a 0.1 micrometer filter membrane into the peak inlet filter after the pump to reduce particle noise from the solvent. After disassembling, clean the small channel for AF4 with millEq water. Hydrate a new regenerated cellulose membrane with a molecular weight cut off of 30 kilodaltons and place the membrane on top of the fret with the shiny side up.Place a wide 350 micrometer spacer below the top half of the channel and assemble the channel parts. Using a torque screwdriver, tighten the screws first to a torque of five Newton meters, then to a torque of seven Newton meters. Tightening the screws around the block in a crisscross pattern.Connect the channels inlet, outlet, and cross flow port to the eclipse. Then from at least three old samples to equilibrate the membrane. Program a fractionation run with the detector flow and focus flow set to one milliliter per minute and an inject flow of 0.2 milliliters per minute.After a one minute pre-focus step, inject the sample over a period of 10 minutes in the focus and inject step, before decreasing the cross flow from two milliliters per minute to 0.1 milliliters per minute over the course of eight minutes. Finish the fractionation run with an elution step without cross flow for 10 minutes and add a washing step of elution and injection, followed by an elution. Then equilibrate the channel for the next run with an initial cross flow of two milliliters per minute and set the UV detector to 280 nanometers.When all of the programs have been set, inject 300 microliters of the sample and record the UV and light scattering signals in the ASTRA software. After 12 and a half minutes, begin collecting one milliliter fractions until 27 minutes post injection. Then perform a glucuronidase assay and a nano particle tracking analysis as demonstrated.Here, the particle recovery and the size of the extracellular vesicles isolated from human vascular endothelial cells after seven days of storage under different conditions are shown in comparison with a fresh sample. Vesicles were subsequently subjected to AF4 purification and the glucuronidase activity per particle was measured. Both size exclusion chromatography and AF4 are successful in separating extracellular vesicle from free glucuronidase.Due to the higher degree of separation for AF4 between particles and free enzyme, contamination of the fractions containing vesicles is less probable. AF4 purification after storage removes free enzyme from the samples and thus lowers the amount of recovered enzyme activity per particle. This effect is most prominent after storage at four degrees Celsius, for which a 2/3 reduction is observed.Omitting this additional purification step can lead to wrong assumption about the enzyme stability. Transmission electron microscopy does not reveal big differences in shape for extracellular vesicles lyophilized without a cryoprotectant. Scanning electron microscopy imaging, however, reveals the presence of aggregates within the lyophilized samples that are not found in the minus 80 degrees stored samples.It is crucial that free enzyme is removed from the vesicles before the glucuronidase assay. Thus, the technique used for vesicle purification needs to be thoroughly evaluated. The purified particles could also be looked at in terms of their surface charge to find out whether the storage changed the natural composition of the membrane proteins or sugars.With our technique, it is possible to get a first indication about extracellular vesicle storage stability, even when there are no known specific markers or assays for the activity available.

evaluation-of-the-storage-stability-of-extracellular-vesicles

Research

JoVE Journal

Bioengineering

13.7K visualizações.  Biogenic Nanotherapeutics group (BION). A estabilidade de armazenamento de vesículas celulares extras é um parâmetro importante para seu uso terapêutico prospectivo. Nosso protocolo fornece uma ferramenta prontamente aplicável para avaliar como o armazenamento afeta as vesículas. A principal vantagem de nossa técnica é que introduzindo glucuronidase como um marcador exógeno, vesículas extracelulares derivadas de diferentes células-mãe são facilmente comparadas com relação à sua armazenamento.O fracionamento d...