Name: evaluation of the storage stability of extracellular vesicles
Uploaded: 2019-05-22T14:30:00
Description: Watch this Scientific Journal Video about Evaluation of the Storage Stability of Extracellular Vesicles at JoVE.com

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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+and+Storage+Stability+of+Extracellular+Vesicles+for+Therapeutic+Applications.">Preservation and Storage Stability of Extracellular Vesicles for Therapeutic Applications.</a> The AAPS Journal. 20 (1), 1 (2017).</li><li>L&#337;rincz, &. #. 1. 9. 3. ;. 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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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">
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		<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 ...

Research

JoVE Journal

Bioengineering

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up ...

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...

Labeling of Extracellular Vesicles for Monitoring Migration and Uptake in Cartilage Explants

Extracellular vesicles (EVs) are used in different studies to prove their potential as a cell-free treatment due to their cargo derived from their ...

Platelet-Derived Extracellular Vesicle Functionalization of Ti Implants

Extracellular Vesicles (EVs) are biological nanovesicles that play a key role in cell communication. Their content includes active biomolecules such as ...