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

德国联邦教育和研究部的 NanoMatFutur 初级研究方案 (赠款编号 13XP5029 a) 支持这项工作。Maximilian Richter 由德国学术奖学金基金会 (Studienstiftung des Deutschen Volkes) 通过博士奖学金提供支助。

1. 细胞培养和细胞调节培养基的生产
<ol><li>一般情况下, 在各自细胞系所需的个别条件下培养细胞。</li>
	<li>在无血清条件下或含有 EV-depleted 耗尽的胎儿牛血清 (FBS) 中培养24-72 小时的细胞。 注: 如果使用 EV-D20E fbs, 请采用一种经证明可以有效耗尽血清的方法, 以防止牛血清衍生的 Ev26污染。</li>
	<li>从烧瓶中收集介质。以 300 x g离心10分钟将细胞颗粒状。小心收集细胞条?...

<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. 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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. 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D., Shah, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+isolating+extracellular+vesicles+impact+down-stream+analyses+of+their+cargoes.">Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes.</a> Methods. 87, 3-10 (2015).</li><li>Patel, D. 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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在这篇手稿中, 我们提出了一个全面的协议, 研究在不同的存储条件下的电动车的稳定性。该协议结合了封装的葡萄糖苷酶作为功能读数, 并对电动车的物理化学参数进行了评估, 从而可以对电动车进行简单的存储稳定性评估, 并对不同细胞的电动车进行比较线。SEM 和 TEM 作为互补方法, 可以深入了解 Ev 在单粒子水平上的变化。这里给出的结果表明, 由于没有低温保护剂的冻干, 电动车和脂质体有聚?...

Ev 是由几乎所有细胞类型产生的膜结合纳米粒子。对于哺乳动物细胞, ev 可以细分为两个主要组, 具有不同的生产途径1,2。膜囊泡的大小范围约为100-1000 纳米, 是通过细胞膜的直接萌发而产生的。外质体, 大小30-200 纳米, 是从由内芽形成的多囊体形成的外质体, 随后与细胞膜融合, 同时释放多个外质体。这些囊泡的主要功能是细胞3之间的信息传输。为此, 对 RNA、DNA 和蛋白质等货物进行了积极的分类。电动车可以对其目标传递各种影响, 对健康和疾病状态都有影响。一方面, 他们介导阳性效应, 如组织再生, 抗原表达, 或抗生素的影响, 使他们的发展吉祥的目标, 作为治疗方法4,5。另一方面, Ev 能促进肿瘤血管化6, 诱导旁观者在应激反应中的作用 7, 并可能在自身免疫性疾病8和炎症性疾病9中起到一定作用。因此, 它们可能是更好地了解许多病理影响的关键组成部分。然而, 在癌症10、11、12 和心血管疾病13等多种疾病中, 发生了改变后的电动车, 而且在血液和尿液中容易获得这些疾病, 使其成为理想的生物 标志 物。最后, 它们良好的生物相容性14和固有的靶向能力使电动车对药物输送也很有趣。在这篇手稿中, 我们描述了一个评估从哺乳动物细胞中提取的 Ev 的存储稳定性的协议, 这是一个重要的性质, 目前还很少被研究。
对于电动车的临床发展, 仍有许多障碍要克服 16, 包括对其治疗效果的评价、生产、净化和储存1 7。虽然-80°c 被广泛视为 EV 存储18的黄金标准, 但所需的冰柜成本很高, 从生产到患者, 维护所需的冷链可能具有挑战性。此外, 一些报告表明, 在-80°c 下的存储仍然不能最佳地保留电动车, 并导致 ev 功能的损失19,20。其他方法, 如冷冻干燥21,22或喷雾干燥 23, 已被提出作为潜在的替代品冷冻储存的电动车。
评估储存稳定性的最佳方法是测试功能检测中的 ev, 或通过评估特定标记 (例如, 其抗菌活性19) 来测试 ev。当知道水泡的预期效果, 当一个不同的一组电动车要研究时, 这是可能的。如果要比较来自不同细胞源的 Ev (例如, 用于药物封装), 或者没有已知的功能读数, 则无法再直接评估由于存储而产生的变化。
另一方面, 简单地评估其物理化学参数的变化, 如大小、颗粒恢复和蛋白质浓度, 并不总是预测 EV 活性的变化, 最近的专利20就表明了这一点。
在这里, 我们提供了一个易于应用的协议, 通过评估其物理化学参数与封装的β-葡萄糖酶酶作为电动车货物的替代品的活性来测量其储存稳定性。酶的负荷是通过皂甙的培养完成的, 这是一种用来自不同来源21、24、25 的电动车建立的温和方法。皂甙在 EV 膜中形成短暂的毛孔, 使酶被吸收到囊泡中。由于酶在不受储存条件的影响时容易失去活性, 它们是评估电动车功能货物保存情况的理想替代品。
我们已经证明, 该方案的应用对来自人间充质干细胞 (MSCs)、人脐静脉内皮细胞 (Huvec) 和人腺癌肺泡上皮细胞 (A549) 的 Ev 确实导致了巨大的差异。不同细胞系之间的存储稳定性, 在选择 EV 源21时应考虑到这一点。

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			<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>
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			<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

细胞外囊泡 (Ev) 是目前研究的目标, 可用作药物、药物载体和生物标志物。对于他们的临床发展, 不仅他们的药物活性很重要, 他们的生产也需要评估。在此背景下, 研究的重点是电动车的隔离、特性和存储。本手稿旨在提供一个简单的程序, 以评估不同的存储条件对 Ev 的影响, 而无需基因操纵或特定的功能检测。这样就可以快速获得在给定存储条件下电动车稳定性的第一印象, 并且可以很容易地比较来自不同细胞源的电动车。稳定性测量是基于 Ev 的物理化学参数 (大小、颗粒浓度和形态) 和保持其货物的活性。后者是由皂甙介导的酶β-葡萄糖苷酶封装到 Ev 中进行评估的。葡萄糖苷酶作为一个替代品, 并允许一个简单的定量, 通过荧光记者分子的裂解。目前的协议可以成为研究人员寻找最佳保持 EV 特性的存储条件的工具, 以推进 EV 研究的临床应用。

图 1显示了与 huvec 隔离的 ev 的存储特性。用 UC 分离出的电动车, 对葡萄糖苷酶进行了包封, 并在证交会后, 用 NTA 对纯化后的电动车的理化性质进行了评价。随后对囊泡样品进行了 AF4 纯化, 并测定了葡萄糖苷酶活性。
然后将囊泡在4°c 或-80°c 和4°c 以冻干形式储存 7 d, 在后一种情况下, 添加4% 的海藻糖。储存后, 用 NTA 再次测定囊泡, 在 AF4 后, 对每个颗粒剩余的葡?...

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

细胞外囊储存稳定性的评价

在这里, 我们提出了一个易于应用的协议, 以评估细胞外囊泡的储存稳定性, 一组自然发生的纳米颗粒产生的细胞。囊泡中含有葡萄糖苷酶作为模型酶, 并在不同条件下储存。储存后, 对其理化参数和包封酶的活性进行了评价。

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

外细胞囊泡的储存稳定性是细胞囊泡未来治疗使用的重要参数。我们的协议提供了一个易于适用的工具，用于评估存储对囊泡的影响。我们技术的主要优点是，通过将葡萄糖酶作为外源性标记物引入，从不同母细胞中提取的细胞外囊泡很容易比较，可以比较其可耐性。非对称流场流量分馏（AF4）是非常敏感化合物大小排除色谱纯化的温和替代品。因为化合物在通道中遇到的压力较小。由于葡萄糖酶是一种灵敏酶，因此，通过周密的规划和进行自给和背对背的纯化和分析步骤，对粘液酶的质量有好处。根据这些细胞的标准条件培养感兴趣的细胞系后，用无血清或额外的细胞囊泡耗尽介质替换上一提液，将细胞返回到细胞培养箱中再增加24至72小时。在孵化结束时，从每个烧瓶中收集超级纳兰特，并离心样品沉淀细胞。小心地收集细胞调节介质，而不干扰颗粒。并再次离心介质，以清除细胞碎片和大集料。然后小心地将超级纳特转移到超离心管中。如果使用固定角度转子，则标记离心机中管的方向，以便于离心后检索细胞外囊泡颗粒。在超离心结束时，使用血清移液器小心丢弃上流液，而不会干扰细胞外囊泡颗粒。在200微升0.2微米孔隙过滤PBS到第一个超离心管的残余上清液。重新悬浮颗粒后，将产生的细胞外囊泡悬浮液转移到下一管，以便重新悬浮随后的颗粒，直到所有细胞外囊泡颗粒都重新悬浮。将β葡萄糖糖酶加入最终重新暂停的颗粒溶液，最终浓度为每毫升1.5毫克。和皂素到最终浓度0.1毫克每毫升。然后通过涡旋混合三秒钟，将反应在室温下放置10分钟，间歇性轻柔轻拂。一旦葡萄糖酶被封装，一定要在同一天执行所有后续纯化囊泡评估步骤，以获得无偏不倚的储存稳定性结果。将尺寸排除色谱柱与至少两列 PBS 平衡，准备在皂素孵育后立即净化颗粒。要净化，将高达400微升的细胞外囊泡悬浮液加入到柱子上。然后收集一毫升的分馏器。为了符合细胞外囊泡与污染蛋白和自由葡萄糖酶的分离，请根据制造商的说明，通过双辛酸测定测量蛋白质浓度。使用针对相应仪器和囊泡类型的优化参数，使用纳米颗粒跟踪分析仪器测量细胞外囊泡的大小和浓度。为了测量葡萄糖酶活性，在纯化的细胞外囊泡的125微升中加入25微升新鲜制备的荧光素二β-D-葡萄糖酰胺，并将样品添加到黑色96孔板的一个井中。在 480 纳米的激励和 516 纳米的发射下测量板读卡器上零荧光素生产的时间。用透明塑料箔紧紧盖住板，以减少蒸发。然后在37摄氏度下孵育防光板18小时。测量板读卡器上 18 小时荧光素的产生，正如刚刚演示的。对于细胞外囊泡冻干，在纯化的囊泡中每毫升海囊糖中加入四毫克。在零下80摄氏度下将囊泡冷冻至少一小时。要使样品冻干，请将冻干剂的货架温度设置为 15 摄氏度，将压力设置为 0.180 毫巴。在这些条件下干燥囊泡46小时。在主干燥步骤结束时，将货架温度设置为 25 摄氏度，将压力设置为 0035 毫巴，并在这些条件下将样品干燥两小时。然后将冻干样品储存在摄氏四度。要在储存后对样品进行补水，在冻干之前添加一个与细胞外囊泡悬浮液量相等的水量。为了评估储存后酶的活性，将AF4仪器装上新准备的0.1微米过滤PBS，用于移动相，并在泵后将0.1微米滤膜插入峰值进气过滤器，以减少来自溶剂的颗粒噪音。拆卸后，用千禧年水清洁 AF4 的小通道。水合一种新的再生纤维素膜，分子量切断30千吨，将膜放在具有光泽的一面的烦恼上。将宽 350 微米的空间放在通道上半部分下方，然后组装通道部件。使用扭矩螺丝刀，先拧紧螺钉到五牛顿米的扭矩，然后拧紧到 7 牛顿米的扭矩。以纵横交错的图案拧紧块周围的螺钉。将通道入口、出口和交叉流量端口连接到日食。然后从至少三个旧样品平衡膜。在检测器流量和焦点流设置为每分钟 1 毫升和注入流量为每分钟 0.2 毫升时进行分馏运行。经过一分钟的预对焦步骤后，在对焦中注射样品 10 分钟后注入，然后注入步长，然后将交叉流量从每分钟两毫升减小到每分钟 0.1 毫升。用洗脱步骤完成分馏运行，无需交叉流 10 分钟，然后添加洗涤步骤的洗洗和注射，然后是洗脱。然后平衡下一次运行的通道，初始交叉流量为每分钟两毫升，并设置紫外线检测器为 280 纳米。设置所有程序后，注入样品的 300 微升，并在 ASTRA 软件中记录 UV 和光散射信号。12分半钟后，开始收集一毫升分数，直到注射后27分钟。然后进行葡萄糖酶测定和纳米颗粒跟踪分析，如所证明的。在这里，与新鲜样本相比，在不同条件下储存7天后，从人类血管内皮细胞中分离出的颗粒回收和细胞外囊泡的大小显示出来。囊泡随后受到AF4纯化，并测量了每个粒子的葡萄糖酶活性。大小排除色谱和AF4都成功地将细胞外囊泡与自由葡萄糖酶分离。由于AF4在颗粒和自由酶之间的分离程度较高，含有囊泡的馏分污染的可能性较小。储存后AF4纯化会从样品中去除无活性酶，从而降低每个颗粒的恢复酶活性。这种效应在储存在摄氏四度后最突出，观察到2/3的减少。省略此额外的纯化步骤可能会导致对酶稳定性的错误假设。传输电子显微镜不会显示细胞外囊泡在没有低温保护剂的情况下冻干的形状差异。然而，扫描电子显微镜成像揭示了冻干样品中存在聚合，而在储存的负80度样品中却找不到这些聚集体。在葡萄糖酶测定之前，从囊泡中去除自由酶至关重要。因此，需要对用于囊泡纯化的技术进行彻底评估。纯化的颗粒也可以根据其表面电荷来观察，以确定储存是否改变了膜蛋白或糖的自然成分。使用我们的技术，即使没有已知的特定标记或可用的活动检测，也可以获得关于细胞外囊泡储存稳定性的第一个指示。

Research

JoVE Journal

Bioengineering

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

磁共振弹性成像方法评价组织工程构造增长

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

科研

生物工程

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

恒压控制的挤压方法制备纳米脂质体

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

无细胞的转录和翻译的机械囊泡的封装建设模拟蜂窝

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

定量和大小分析外囊泡的使用可调电阻脉冲检测

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

3d轨道跟踪的改进双光子显微镜之应用：细胞内的囊泡的跟踪

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

基于纸张的设备进行隔离外囊泡与表征

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

细胞外基质衍生的泡沫和微载体如组织特异性细胞培养和交付平台的制造

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

使用声学分离器对CHO收获的细胞培养液进行初步澄清

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

标记细胞外囊泡以监测软骨外植体的迁移和摄取

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

血小板衍生的Ti植入物的细胞外囊泡功能化

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