细胞外囊储存稳定性的评价
Summary
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。
Abstract
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。
Introduction
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。
Protocol
1. 细胞培养和细胞调节培养基的生产 一般情况下, 在各自细胞系所需的个别条件下培养细胞。 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。 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?…
Representative Results
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?…
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 1,2 dimyristoyl-sn glycero-3-phospho-choline (DMPC) | Sigma-Aldrich | P2663-25MG | |
| 1,2-dipalmitoyl-sn-glycero-3-phospho-choline (DPPC) | Sigma-Aldrich | P4329-25MG | |
| 225 cm² cell culture flasks | Corning | 431082 | Used with 25 ml of medium |
| 30 kDa regenerated cellulose membrane | Wyatt Technology Europe | 1854 | |
| 350 µm spacer | Wyatt Technology Europe | ||
| Automated fraction collector | Thermo Fisher Scientific | ||
| Beta-glucuronidase | Sigma-Aldrich | G7646-100KU | |
| Chloroform | Fisher scientific | C/4966/17 | |
| Column oven | Hitachi High-Technologies Europe | ||
| D-(+)-Trehalose dihydrate | Sigma-Aldrich | T9531-10G | |
| DAWN HELEOS II, Multi-angle light scattering detector | Wyatt Technology Europe | ||
| Durapore Membrane filter, PVDF, 0,1 µm, 47 mm | Merck | VVLP04700 | Used for the preparation of buffers for AF4 |
| EBM-2 | Lonza Verviers, S.p.r. | CC-3156 | Endothelial Cell Growth basal medium, used for the serum free culture of HUVEC cells |
| Eclipse dualtec | Wyatt Technology Europe | ||
| EGM-2 | Lonza Verviers, S.p.r. | CC-3162 | Endothelial Cell Growth medium, used for the normal culture of HUVEC cells |
| ELISA Plate Sealers | R&D Systems | DY992 | used for sealing of 96-well plates for the glucuronidase assay |
| Ethanol | Fisher scientific | E/0665DF/17 | |
| Extruder Set With Holder/Heating Block | Avanti Polar Lipids | 610000-1EA | |
| Filter support | Avanti Polar Lipids | 610014-1EA | used for liposome preparation |
| Fluorescein di-β-D-glucoronide | Thermo Fisher Scientific | F2915 | |
| Gibco PBS-tablets+CA10:F36 | Thermo Fisher Scientific | 18912014 | |
| Hettich Universal 320 R | Andreas Hettich GmbH & Co.KG | Used for pelleting cells at 300 g | |
| Hettich Rotina 420 R | Andreas Hettich GmbH & Co.KG | Used for pelleting larger debris at 3000 g | |
| HUVEC cells | Lonza Verviers, S.p.r. | C2517A | |
| Kimble FlexColumn 1X30CM | Kimble | 420401-1030 | |
| Lyophilizer ALPHA 2-4 LSC | Christ | ||
| Microcentrifuge Tubes, Polypropylene | VWR international | 525-0255 | the tubes used for all EV-handling, found to be more favorable than comparable products from other suppliers regarding particle recovery |
| Nanosight LM14 equipped with a green laser | Malvern Pananalytical | ||
| Nanosight-software version 3.1 | Malvern Pananalytical | ||
| Nucleopore 200 nm track-etch polycarbonate membranes | Whatman/GE Healthcare | 110406 | used for liposome preparation |
| PEEK Inline filter holder | Wyatt Technology Europe | ||
| Phosphotungstic acid hydrate | Sigma-Aldrich | 79690-25G | |
| Polycarbonate bottles for ultracentrifugation | Beckman Coulter | 355622 | |
| QuantiPro BCA Assay Kit | Sigma-Aldrich | QPBCA-1KT | |
| Saponin | Sigma-Aldrich | 47036 | |
| Scanning electron microscopy Zeiss EVO HD 15 | Carl Zeiss AG | ||
| Sepharose Cl-2b | GE Healthcare | 17014001 | |
| SEM copper grids with carbon film | Plano | S160-4 | |
| Small AF4 channel | Wyatt Technology Europe | ||
| Sputter-coater Q150R ES | Quorum Technologies | ||
| Transmission electron microscopy JEOL JEM 2011 | Oxford Instruments | ||
| Type 45 Ti ultracentrifugation rotor | Beckman Coulter | 339160 | |
| Ultimate 3000 Dionex autosampler | Thermo Fisher Scientific | ||
| Ultimate 3000 Dionex isocratic pump | Thermo Fisher Scientific | ||
| Ultimate 3000 Dionex online vacuum degasser | Thermo Fisher Scientific | ||
| Ultracentrifuge OptimaTM L-90 K | Beckman Coulter | ||
| UV detector | Thermo Fisher Scientific | ||
| Whatman 0.2 µm pore size mixed cellulose filter | Whatman/GE Healthcare | 10401712 | Used for the filtration of all buffers used with the EVs and in SEC |
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Cite This Article
Richter, M., Fuhrmann, K., Fuhrmann, G. Evaluation of the Storage Stability of Extracellular Vesicles. J. Vis. Exp. (147), e59584, doi:10.3791/59584 (2019).