利用超滤离心技术分离小鼠支气管肺泡灌洗液中的细胞外囊
Summary
在这里, 我们描述了两个细胞外囊泡分离方案, 超滤离心和超离心与密度梯度离心, 从小鼠支气管肺泡灌洗液样品中分离细胞外囊泡。用两种方法对小鼠支气管肺泡灌洗液中的细胞外囊泡进行了定量和表征。
Abstract
细胞外囊泡 (ev) 是新发现的亚细胞成分, 在生理和病理状态下的许多生物信号功能中起着重要作用。由于每种技术固有的限制, ev 的隔离仍然是这一领域的主要挑战。采用密度梯度离心法的微分超离心法是一种常用的方法, 被认为是电动汽车隔离的黄金标准程序。然而, 此过程是耗时的, 劳动密集型的, 一般会导致低可扩展性, 这可能不适合小体积样品, 如支气管肺泡灌洗液。证明了超滤离心分离方法简单、时间高效、省力, 但具有较高的回收率和纯度。我们建议这种隔离方法可以是一种替代方法, 适用于电动汽车隔离, 特别是小批量生物样品。
Introduction
外质体是 ev 的最小子集, 直径为 50–200 nm, 在各种信令过程1、2、3、4、5中具有多种生物功能。它们主要通过通过脂类、蛋白质和核酸等货物分子促进细胞间的交流来控制细胞和组织的稳态.电动汽车研究的一个关键步骤是隔离过程。有或没有密度梯度离心 (dgc) 的差动超离心 (uc) 被认为是黄金标准法, 但这种方法存在很大的局限性, 包括 ev 回收率低和可扩展性低 10,11,12、将其最佳利用限制在较大体积的样品中, 如细胞培养上清液或高外体细胞生产样品。其他方法的优点和缺点, 如超滤或色谱仪排除尺寸、珠子或柱的免疫亲和力分离以及微流体, 都有很好的描述, 现代补充程序已经发展到克服并最大限度地减少每种方法中的技术限制 11、12、13、14、15。其他研究表明, 在过滤单元中使用纳米多孔膜的超滤离心 (ufc) 是一种替代技术, 可提供与 uc 方法16、17、18 相当的纯度。这种技术可以被认为是一种替代的隔离方法。
支气管肺泡灌洗液 (balf) 含有在不同呼吸条件下具有多种生物功能的ev19、20、21、22.研究 bal 衍生的 ev 带来了一些挑战, 由于在人类的支气管镜检查程序的侵入性, 以及有限的大量灌洗液回收。在小动物, 如小鼠, 只有几毫升可以恢复在正常的肺条件下, 更不知道在发炎或纤维化的肺 23。因此, 收集足够数量的 balf, 用于通过差动超离心进行电动汽车隔离, 用于下游应用可能是不可行的。然而, 分离正确的 ev 种群是研究电动汽车生物学功能的关键因素。在完善的电动汽车隔离方法中, 效率和功效之间的微妙平衡仍然是一个挑战。
在目前的研究中, 我们证明了一种离心超滤方法, 利用 100 kda 分子量截止 (mwco) 纳米膜过滤单元, 适用于小体积生物样品, 如 balf。该技术简单、高效, 具有较高的纯度和可扩展性, 可支持对 balf 衍生 ev 的研究。
Protocol
动物的利用和所有动物程序得到了塞达西奈医疗中心 (csmc) 机构动物护理和使用委员会 (iacuc) 的批准。 1. 小鼠支气管肺泡灌洗液 (valf) 的采集和制备 balf 系列 用氯胺酮 (300 mgkg) 和 xylazine (30 mg kg) 的鸡尾酒对小鼠进行安乐死, 然后通过腹膜内途径进行颈椎脱位。 将 22 g 血管造影剂插入气管。将含有1毫升 (ml) 冰凉的杜尔贝科磷酸盐缓冲液盐水 (dpbs) 的胰岛素?…
Representative Results
我们在同一天使用 ufc 和 uc-dgc 隔离方法从鼠标 balf 执行 ev 隔离。ufc 方法大约需要 2.5-3小时, 而 uc-dgc 技术需要8小时的处理时间。这不包括缓冲区和试剂制备时间。应当指出, 其他一些任务可以在长离心期内执行。然而, 整个过程持续了近一整天的 uc-dgc 隔离技术。 与 uc-dgc ev (17.67±7.8 nm,图 1b) 相比, ufc 方?…
Discussion
在过去的几十年里, 科学家们已经解开了 ev 在细胞稳态中的意义。更重要的是, ev 通过生物活性货物分子 1、21、22、26、27调节相邻和远的细胞, 在许多疾病过程中发挥着重要作用,28,29,30. 这一领域?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了 nhlbi龙湾国家卫生研究院颁发 hl103868 (至 p. c.) 和 hl137076 (至 p. c.)、美国心脏协会援助奖 (至 p. c.) 和塞缪尔·奥辛综合癌症研究协会 (pc) 的支持。我们要对塞达西奈医疗中心的施密特心脏研究所表示高度赞赏, 该研究所为我们提供了一台用于电动车纳米粒子跟踪分析的纳米视觉机器。
Materials
| Material | |||
| Amicon Ultra-15 centrifugal filters Ultracel-100K | Sigma-Millipore, St. Louis, MO | UFC910024 | |
| Dulbecco's Phosphate Buffered Saline (DPBS) | Corning Cellgro, Manassas, VA | 21-031-CV | |
| Sucrose | Sigma-Millipore, St. Louis, MO | EMD8550 | |
| HEPES | Research Products International, Prospect, IL | 75277-39-3 | |
| EDTA | Corning Cellgro, Manassas, VA | 46-034-CI | |
| Sodium Chloride | Sigma-Millipore, St. Louis, MO | S3014-1KG | |
| OptiPrep | Sigma-Millipore, St. Louis, MO | MKCD9753 | Density Gradient Medium |
| Ketamine | VetOne, Boise, ID | 13985-702-10 | |
| Xylazine | Akorn Animal Health, Lake Forest, IL | 59399-110-20 | |
| Syringe 1 mL | BD Syringe, Franklin Lakes, NJ | 309656 | |
| Angiocatheter 20G | BD Syringe, Franklin Lakes, NJ | 381703 | |
| Centrifuge tubes 15 mL | VWR, Radnor, PA | 89039-666 | |
| Centrifuge tubes 50 mL | Corning Cellgro, Manassas, VA | 430828 | |
| Bicinchonic acid (BCA) protein assay | Pierce, Thermo Fischer Scientific, Rockford, IL | 23235 | |
| Rabbit anti-mouse TSG101 Antibody | AbCam, Cambridge, MA | AB125011 | |
| Rat anti-mouse PE-CD63 Antibody | Biolegend, San Diego, CA | 143904 | |
| CD81 | |||
| CD9 | |||
| Anti-rabbit IgG, HRP-linked antibody | Cell Signaling Technology, Danvers, MA | 7074S | |
| 4x LDS | |||
| 10x Reducing agent (Bolt) | |||
| 10x Lysis buffer (Bolt) | Cell Signaling Technology, Danvers, MA | ||
| Bolt 4-12% Bis-Tris Plus acrylamide gel | Invitrogen, Thermo Fisher Scientific, Waltham, MA | NW04120 | |
| iBlot 2 Nitrocellulose mini stacks | Invitrogen, Thermo Fisher Scientific, Waltham, MA | IB23002 | |
| Chemiluminescent HRP antibody detection reagent HyGLO | Denville Scientific, Holliston, MA | E2400 | |
| Ultracentrifuge tubes 17 mL | Beckman Coulter, Pasadena, CA | 337986 | |
| Ultracentrifuge tubes 38.5 mL | Beckman Coulter, Pasadena, CA | 326823 | |
| Corning SFCA Syringe Filters 0.2 µm pore | Thermo Fisher Scientific, Waltham, MA | 09-754-13 | |
| Equipment | |||
| Centrifuge | Eppendorf, Hamburg, Germany | – | |
| Ultracentrifuge | Beckman Coulter, Pasadena, CA | – | |
| Nanosight (NS300) | Malvern, Worcestershire, UK | – | To measure particle size distribution and particle concentration |
| MACSQuant Analyzer 10 flow cytometer | Miltenyi Biotec, Bergisch Gladbach, Germany | – | |
| iBlot Transfer Apparatus | Thermo Fischer Scientific, Waltham, MA | – | |
| Bio-Rad ChemiDoc MP Imaging System | Bio-Rad, Hercules, CA | ||
| FlowJo v. 10 | Analysis software |
References
- Thery, C., Zitvogel, L., Amigorena, S. Exosomes: composition, biogenesis and function. Nature Reviews Immunology. 2, 569-579 (2002).
- Kosaka, N., et al. Secretory Mechanisms and Intercellular Transfer of MicroRNAs in Living Cells. Journal of Biological Chemistry. 285 (23), 17442-17452 (2010).
- Raposo, G., Stoorvogel, W. Extracellular vesicles: Exosomes, microvesicles, and friends. The Journal of Cell Biology. 200 (4), 373-383 (2013).
- Fujita, Y., Kosaka, N., Araya, J., Kuwano, K., Ochiya, T. Extracellular vesicles in lung microenvironment and pathogenesis. Trends in Molecular Medicine. 21 (9), 533-542 (2015).
- Kalluri, R. The biology and function of exosomes in cancer. Journal of Clinical Investigation. 126 (4), 1208-1215 (2016).
- Janowska-Wieczorek, A., et al. Microvesicles derived from activated platelets induce metastasis and angiogenesis in lung cancer. International Journal of Cancer. 113 (5), 752-760 (2005).
- Valadi, H., et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nature Cell Biology. 9 (6), 654-659 (2007).
- Colombo, M., Raposo, G., Théry, C. Biogenesis, Secretion, and Intercellular Interactions of Exosomes and Other Extracellular Vesicles. Annual Review of Cell and Developmental Biology. 30 (1), 255-289 (2014).
- Rocco, G. D., Baldari, S., Toietta, G. Exosomes and other extracellular vesicles-mediated microRNA delivery for cancer therapy. Translational Cancer Research. 6 (Supplement 8), S1321-S1330 (2017).
- Peterson, M. F., Otoc, N., Sethi, J. K., Gupta, A., Antes, T. J. Integrated systems for exosome investigation. Methods. 87 (1), 31-45 (2015).
- Xu, R., Greening, D. W., Zhu, H. J., Takahashi, N., Simpson, R. J. Extracellular vesicle isolation and characterization: toward clinical application. Journal of Clinical Investigation. 126, 1152-1162 (2016).
- Gardiner, C., et al. Techniques used for the isolation and characterization of extracellular vesicles: results of a worldwide survey. Journal of Extracellular Vesicles. 5 (1), 32945 (2016).
- Inglis, H. C., et al. Techniques to improve detection and analysis of extracellular vesicles using flow cytometry. Cytometry Part A. 87 (11), 1052-1063 (2015).
- Li, P., Kaslan, M., Lee, S. H., Yao, J., Gao, Z. Progress in Exosome Isolation Techniques. Theranostics. 7 (3), 789-804 (2017).
- Willis, G. R., Kourembanas, S., Mitsialis, S. A. Toward Exosome-Based Therapeutics: Isolation, Heterogeneity, and Fit-for-Purpose Potency. Frontiers in Cardiovascular Medicine. 4, 20389 (2017).
- Lobb, R. J., et al. Optimized exosome isolation protocol for cell culture supernatant and human plasma. Journal of Extracellular Vesicles. 4 (1), 27031 (2015).
- Benedikter, B. J., et al. Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies. Scientific Reports. 7 (1), 15297 (2017).
- Vergauwen, G., et al. Confounding factors of ultrafiltration and protein analysis in extracellular vesicle research. Scientific Reports. 7 (1), 2704 (2017).
- Kesimer, M., et al. Characterization of exosome-like vesicles released from human tracheobronchial ciliated epithelium: a possible role in innate defense. The FASEB Journal. 23 (6), 1858-1868 (2009).
- Torregrosa Paredes, P., et al. Bronchoalveolar lavage fluid exosomes contribute to cytokine and leukotriene production in allergic asthma. Allergy. 67 (7), 911-919 (2012).
- Alipoor, S. D., et al. Exosomes and Exosomal miRNA in Respiratory Diseases. Mediators of Inflammation. 2016, 5628404 (2016).
- Hough, K. P., Chanda, D., Duncan, S. R., Thannickal, V. J., Deshane, J. S. Exosomes in Immunoregulation of Chronic Lung Diseases. Allergy. 72 (4), 534-544 (2017).
- Van Hoecke, L., Job, E. R., Saelens, X., Roose, K. Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration. Journal of Visualized Experiments. (123), e55398 (2017).
- Minciacchi, V. R., et al. MYC Mediates Large Oncosome-Induced Fibroblast Reprogramming in Prostate Cancer. 癌症研究. 77 (9), 2306-2317 (2017).
- Koliha, N., et al. Melanoma Affects the Composition of Blood Cell-Derived Extracellular Vesicles. Frontiers in Immunology. 7, 581 (2016).
- Thery, C., Ostrowski, M., Segura, E. Membrane vesicles as conveyors of immune responses. Nature Reviews Immunology. 9, 581-593 (2009).
- Camussi, G., Deregibus, M. C., Bruno, S., Cantaluppi, V., Biancone, L. Exosomes/microvesicles as a mechanism of cell-to-cell communication. Kidney International. 78 (9), 838-848 (2010).
- Lee, Y., El Andaloussi, S., Wood, M. J. Exosomes and microvesicles: extracellular vesicles for genetic information transfer and gene therapy. Human Molecular Genetics. 21, R125-R134 (2012).
- Villarroya-Beltri, C., Baixauli, F., Gutiérrez-Vázquez, C., Sánchez-Madrid, F., Mittelbrunn, M. Sorting it out: Regulation of exosome loading. Seminars in Cancer Biology. 28, 3-13 (2014).
- Hoshino, A. Tumour exosome integrins determine organotropic metastasis. Nature. 527, 329-335 (2015).
- Liu, F., et al. The Exosome Total Isolation Chip. ACS Nano. 11 (11), 10712-10723 (2017).
- Cheruvanky, A., et al. Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator. American Journal of Physiology-Renal Physiology. 292 (5), F1657-F1661 (2007).
- Zhao, Z., Yang, Y., Zeng, Y., He, M. A Microfluidic ExoSearch Chip for Multiplexed Exosome Detection Towards Blood-based Ovarian Cancer Diagnosis. Lab on a Chip. 16 (3), 489-496 (2016).
- Fang, S., et al. Clinical application of a microfluidic chip for immunocapture and quantification of circulating exosomes to assist breast cancer diagnosis and molecular classification. PloS ONE. 12 (4), e0175050 (2017).
- Cheruvanky, A., et al. Rapid isolation of urinary exosomal biomarkers using a nanomembrane ultrafiltration concentrator. American Journal Physiology-Renal Physiology. 292 (5), F1657-F1661 (2007).
- Kornilov, R., et al. Efficient ultrafiltration-based protocol to deplete extracellular vesicles from fetal bovine serum. Journal of Extracellular Vesicles. 7 (1), 1422674 (2018).
- Alvarez, M. L., Khosroheidari, M., Kanchi Ravi, R., DiStefano, J. K. Comparison of protein, microRNA, and mRNA yields using different methods of urinary exosome isolation for the discovery of kidney disease biomarkers. Kidney International. 82 (9), 1024-1032 (2012).
- Bosch, S., et al. Trehalose prevents aggregation of exosomes and cryodamage. Scientific Reports. 6 (1), 329 (2016).
- Xiao, J., et al. Cardiac progenitor cell-derived exosomes prevent cardiomyocytes apoptosis through exosomal miR-21 by targeting PDCD4. Cell Death & Disease. 7 (6), e2277 (2016).
- Agarwal, U., et al. Experimental, Systems and Computational Approaches to Understanding the MicroRNA-Mediated Reparative Potential of Cardiac Progenitor Cell-Derived Exosomes From Pediatric Patients. Circulation Research. 120 (4), 701-712 (2017).
- Merchant, M. L., et al. Microfiltration isolation of human urinary exosomes for characterization by MS. PROTEOMICS – Clinical Applications. 4 (1), 84-96 (2010).
- Gouin, K., et al. A comprehensive method for identification of suitable reference genes in extracellular vesicles. Journal of Extracellular Vesicles. 6 (1), 1347019 (2017).
- Betsuyaku, T., et al. Neutrophil Granule Proteins in Bronchoalveolar Lavage Fluid from Subjects with Subclinical Emphysema. American Journal of Respiratory and Critical Care Medicine. 159 (6), 1985-1991 (1999).
Cite This Article
Parimon, T., Garrett III, N. E., Chen, P., Antes, T. J. Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique. J. Vis. Exp. (141), e58310, doi:10.3791/58310 (2018).