基于微流控技术的外泌体分离方法的研究进展
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摘要
外泌体是一种由细胞分泌的,直径一般为30-150 nm的囊泡。外泌体携带有多种蛋白质、mRNA及miRNA等生物标记物,并直接参与细胞间的信息传递、抗原传递、蛋白转运以及RNA转录等重要的生命活动过程,与癌症等多种疾病的发生密切相关,因此在疾病的发生机制探索和相关疾病的检测中具有重大的应用价值。然而,外泌体通常以游离的形式存在于体液中,对外泌体的分离和纯化是实现基于外泌体的疾病发生机制及疾病检测应用研究的基础。近年来,研究人员利用外泌体的生物物理和生物化学性质研发了多种分离和纯化外泌体的方法技术,主要有超速离心法、聚合物沉淀法、免疫分离法以及基于微流控的分离法等。综述了近年来外泌体分离和纯化方法的研究进展,简要论述了传统的外泌体分离方法,重点介绍了基于微流控技术的外泌体分离方法,并比较了这些方法的分离机制、优缺点以及应用前景。通过对近年来外泌体分离和纯化方法的研究现状进行归纳和比较,旨为相关研究人员开展外泌体研究工作提供参考,从而进一步推进外泌体在疾病检测及其他生物医学应用的研究进展。
Exosomes are nanoscale extracellular vesicles of diameter 30-150 nm secreted by cells. Exosomes contain many specificbiomarkers such as proteins,mRNA,miRNA,and participate in many important biological processes such as intercellular communications,antigen presentation,and the transport of proteins,RNA,and other molecules. Exosomes are also associated with the occurrence of cancerand other diseases. Therefore,exosomes have significant application value in the investigation of disease mechanism,disease detection anddiagnostics. However,these exosomes-based applications rely on the isolation and purification of exosomes because they are generally dispersedin body fluids. In recent several years,based on their physical or chemical properties many methods and techniques for the isolation andpurification of exosomes have been developed,such as ultracentrifugation,polymer precipitation,immunoaffinity capture,and microfluidicsbased methods. This article reviewed recent advances in methods and techniques for exosomes isolation and purification,including the briefintroduction of conventional exosomes isolation methods,the detailed introduction of microfluidic-based exosomes isolation techniques,and the contrast of these methods in the mechanism,performance,and application prospects. By inducing and comparing recent advancesin exosome isolation and purification,this review aims to provide a reference for the researchers in exosomes-related research work,andindirectly promote the exosomes research in disease detection and other biomedical applications.
Exosomes are nanoscale extracellular vesicles of diameter 30-150 nm secreted by cells. Exosomes contain many specificbiomarkers such as proteins,mRNA,miRNA,and participate in many important biological processes such as intercellular communications,antigen presentation,and the transport of proteins,RNA,and other molecules. Exosomes are also associated with the occurrence of cancerand other diseases. Therefore,exosomes have significant application value in the investigation of disease mechanism,disease detection anddiagnostics. However,these exosomes-based applications rely on the isolation and purification of exosomes because they are generally dispersedin body fluids. In recent several years,based on their physical or chemical properties many methods and techniques for the isolation andpurification of exosomes have been developed,such as ultracentrifugation,polymer precipitation,immunoaffinity capture,and microfluidicsbased methods. This article reviewed recent advances in methods and techniques for exosomes isolation and purification,including the briefintroduction of conventional exosomes isolation methods,the detailed introduction of microfluidic-based exosomes isolation techniques,and the contrast of these methods in the mechanism,performance,and application prospects. By inducing and comparing recent advancesin exosome isolation and purification,this review aims to provide a reference for the researchers in exosomes-related research work,andindirectly promote the exosomes research in disease detection and other biomedical applications.
引文
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[28]Ibsen SD, Wright J, Lewis JM, et al. Rapid isolation and detectionof exosomes and associated biomarkers from plasma[J]. ACSNano, 2017, 11(7):6641-6651.
[29]Sonnenberg A, Marciniak JY, Krishnan R, et al. DielectrophoreticisolationofDNAandnanoparticlesfromblood[J].Electrophoresis, 2012, 33(16):2482-2490.
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[31]Chao L, Xue C, Chen X, et al. Size-based separation of particles andcells utilizing viscoelastic effects in straight microchannels[J].Anal Chem, 2015, 87(12):6041-6048.
[32]Yuan D, Zhang J, Sluyter R, et al. Continuous plasma extractionunder viscoelastic fluid in a straight channel with asymmetricalexpansion-contraction cavity arrays[J]. Lab Chip, 2016, 16(20):3919-3928.
[33]Zhang J, Yan S, Yuan D, et al. A novel viscoelastic-based ferrofluidfor continuous sheathless microfluidic separation of nonmagneticmicroparticles[J]. Lab Chip, 2016, 16(20):3947-3956.
[34]Liu C, Guo J, Tian F, et al. Field-free isolation of exosomes fromextracellular vesicles by microfluidic viscoelastic flows[J]. ACSNano, 2017, 11(7):6968-6976.
[35]Wunsch BH, Smith JT, Gifford SM, et al. Nanoscale Lateraldisplacement arrays for the separation of exosomes and colloidsdown to 20 nm[J]. Nat Nanotechnol, 2016, 11(11):936.
[36]Davies RT, Kim J, Jang SC, et al. Microfluidic filtration system toisolate extracellular vesicles from blood[J]. Lab Chip, 2012, 12(24):5202-5210.
[37]Yasui T, Yanagida T, Ito S, et al. Unveiling massive numbers ofcancer-related urinary-microRNA candidates via nanowires[J].Science Advances, 2017, 3(12):e1701133.
[38]He M, Crow J, Roth M, et al. Integrated immunoisolation andprotein analysis of circulating exosomes using microfluidictechnology[J]. Lab Chip, 2014, 14(19):3773-3780.
[39]Chiu YJ, Cai W, Shih YR, et al. A single-cell assay for time Lapsestudies of exosome secretion and cell behaviors[J]. Small, 2016,12(27):3658-3666.
[40]Deun JV, Mestdagh P, Sormunen R, et al. The impact of disparateisolation methods for extracellular vesicles on downstream RNAprofiling[J]. J Extracell Vesicles, 2014, 3(1):24858.
[41]Linares R, Tan S, Gounou C, et al. High-speed centrifugationinduces aggregation of extracellular vesicles[J]. J ExtracellVesicles, 2015, 4(1):29509.
[42]YangF,LiaoX, TianY, etal.Exosomeseparationusingmicrofluidic systems:size-based, immunoaffinity-based anddynamic methodologies[J]. Biotechnol J, 2017, 12(4):160069.
[2]Mahaweni NM, Kaijenlambers MEH, Dekkers J, et al. Tumourderived exosomes as antigen delivery carriers in dendritic cellbased immunotherapy for malignant mesothelioma[J]. J ExtracellVesicles, 2013, 2. doi:10.3402/jev.v210.22492.
[3]Buzas EI, Gy Rgy B, Nagy G, et al. Emerging role of extracellularvesiclesininflammatorydiseases[J].NatureReviewsRheumatology, 2014, 10(6):356-364.
[4]Gallo A, Tandon M, Alevizos I, et al. The majority of microRNAsdetectable in serum and saliva is concentrated in exosomes[J].PLoS One, 2012, 7(3):e30679.
[5]Whiteside TL. Exosomes carrying immunoinhibitory proteins andtheir role in cancer[J]. Clinical and Experimental Immunology,2017, 189(3):259-267.
[6]Youhei Tanaka MD, Hidenobu Kamohara MDPhD, et al. Clinicalimpact of serum exosomal microRNA-21 as a clinical biomarker inhuman esophageal squamous cell carcinoma[J]. Cancer, 2013,119(6):1159-1167.
[7]Kalluri R. The biology and function of exosomes in cancer[J]. JClin Invest, 2016, 126(4):1208-1215.
[8]Taylor DD, Gercel-Taylor C. MicroRNA signatures of tumorderived exosomes as diagnostic biomarkers of ovarian cancer[J].Gynecologic Oncology, 2008, 110(1):13-21.
[9]Schageman J, Zeringer E, Li M, et al. The complete exosomeworkflow solution:from isolation to characterization of RNACargo[J]. Biomed Res Int, 2013, 2013(8):253957.
[10]邢宇洋,康亚妮,戚颖.乳腺癌细胞外泌体的分离与鉴定[J].肿瘤, 2013, 33(7):581-584, 596.
[11]陈加贵,邓敬桓,何敏.肝癌细胞外泌体的分离与鉴定[J].世界华人消化杂志, 2016, 5:737-743.
[12]卢婉,杨人强,王伶.外泌体的研究进展[J].生命的化学,2013, 51(4):438-442.
[13]胡国文,李青,牛鑫.旋转超滤:一种提取细胞外泌体的新方法[J].第二军医大学学报, 2014, 35(6)598-602.
[14]Liu F, Vermesh O, Mani V, et al. The Exosome Total IsolationChip[J]. ACS Nano, 2017, 11(11):10712-10723.
[15]Oksvold MP, Neurauter A, Pedersen KW. Magnetic bead-basedisolation of exosomes[J]. Methods in Molecular Biology, 2015,1218:465.
[16]Weng Y, Sui Z, Shan Y, et al. Effective isolation of exosomes withpolyethylene glycol from cell culture supernatant for in-depthproteome profiling[J]. Analyst, 2016, 141(15):4640-4646.
[17]Augustsson P, Karlsen JT, Su HW, et al. Iso-acoustic focusing ofcells for size-insensitive acousto-mechanical phenotyping[J].Nature Communications, 2016, 7:11556
[18]Ding X, Lin SC, et al. On-chip manipulation of single microparticles, cells, and organisms using surface acoustic waves[J]. ProcNatl Acad Sci USA, 2012, 109(28):11105-11109.
[19]Ma Z, Zhou Y, Collins DJ, et al. Fluorescence activated cell sortingvia a focused traveling surface acoustic beam[J]. Lab Chip,2017, 17(18):3176-3185.
[20]Wu M, Ouyang Y, Wang Z, et al. Isolation of exosomes from wholeblood by integrating acoustics and microfluidics[J]. Proc NatlAcad Sci USA, 2017, 114(40):201709210.
[21]Lee K, Shao H, et al. Acoustic purification of extracellularmicrovesicles[J]. ACS Nano, 2015, 9(3):2321-2327.
[22]Feng JJ, Krishnamoorthy S, Sundaram S. Numerical analysisofmixingbyelectrothermalinducedflowinmicrofluidicsystems[J]. Biomicrofluidics, 2007, 1(2):24102.
[23]方明,樊磊,曾一笑.集成阵列叉指电极介电泳芯片粒子分离[J].微纳电子技术, 2016, 53(7):461-466.
[24]任玉坤,敖宏瑞,顾建忠.面向微系统的介电泳力微纳粒子操控研究[J].物理学报, 2009, 58(11):7869-7877.
[25]吴玉潘.基于交流电热的微流体混合芯片研究[D].哈尔滨:哈尔滨工业大学, 2015.
[26]Pohl HA. Dielectrophoresis:the behavior of neutral matter innonuniform electric fields[M]. London:Cambridge UniversityPr, 1978.
[27]Heineck DP, Lewis JM, Heller MJ. Electrokinetic device designand constraints for use in high conductance solutions[J].Electrophoresis, 2017, 38(11):1475-1482.
[28]Ibsen SD, Wright J, Lewis JM, et al. Rapid isolation and detectionof exosomes and associated biomarkers from plasma[J]. ACSNano, 2017, 11(7):6641-6651.
[29]Sonnenberg A, Marciniak JY, Krishnan R, et al. DielectrophoreticisolationofDNAandnanoparticlesfromblood[J].Electrophoresis, 2012, 33(16):2482-2490.
[30]Lu X, Xuan X. Continuous microfluidic particle separation viaelasto-inertial pinched flow fractionation(eiPFF)[J]. AnalChem, 2015, 87(12):6389-6396.
[31]Chao L, Xue C, Chen X, et al. Size-based separation of particles andcells utilizing viscoelastic effects in straight microchannels[J].Anal Chem, 2015, 87(12):6041-6048.
[32]Yuan D, Zhang J, Sluyter R, et al. Continuous plasma extractionunder viscoelastic fluid in a straight channel with asymmetricalexpansion-contraction cavity arrays[J]. Lab Chip, 2016, 16(20):3919-3928.
[33]Zhang J, Yan S, Yuan D, et al. A novel viscoelastic-based ferrofluidfor continuous sheathless microfluidic separation of nonmagneticmicroparticles[J]. Lab Chip, 2016, 16(20):3947-3956.
[34]Liu C, Guo J, Tian F, et al. Field-free isolation of exosomes fromextracellular vesicles by microfluidic viscoelastic flows[J]. ACSNano, 2017, 11(7):6968-6976.
[35]Wunsch BH, Smith JT, Gifford SM, et al. Nanoscale Lateraldisplacement arrays for the separation of exosomes and colloidsdown to 20 nm[J]. Nat Nanotechnol, 2016, 11(11):936.
[36]Davies RT, Kim J, Jang SC, et al. Microfluidic filtration system toisolate extracellular vesicles from blood[J]. Lab Chip, 2012, 12(24):5202-5210.
[37]Yasui T, Yanagida T, Ito S, et al. Unveiling massive numbers ofcancer-related urinary-microRNA candidates via nanowires[J].Science Advances, 2017, 3(12):e1701133.
[38]He M, Crow J, Roth M, et al. Integrated immunoisolation andprotein analysis of circulating exosomes using microfluidictechnology[J]. Lab Chip, 2014, 14(19):3773-3780.
[39]Chiu YJ, Cai W, Shih YR, et al. A single-cell assay for time Lapsestudies of exosome secretion and cell behaviors[J]. Small, 2016,12(27):3658-3666.
[40]Deun JV, Mestdagh P, Sormunen R, et al. The impact of disparateisolation methods for extracellular vesicles on downstream RNAprofiling[J]. J Extracell Vesicles, 2014, 3(1):24858.
[41]Linares R, Tan S, Gounou C, et al. High-speed centrifugationinduces aggregation of extracellular vesicles[J]. J ExtracellVesicles, 2015, 4(1):29509.
[42]YangF,LiaoX, TianY, etal.Exosomeseparationusingmicrofluidic systems:size-based, immunoaffinity-based anddynamic methodologies[J]. Biotechnol J, 2017, 12(4):160069.