[EXO]提取方法比较 ——如何获得你所想要的exosomes？

exosome提取方法比较 ——如何获得你所想要的exosomes？

    外泌体（Exosome）发现于1986年，是一种直径约30~100nm的双层膜囊泡状结构小体，可由机体内多种细胞如免疫细胞、干细胞、心血管细胞、网织红细胞、血小板、神经细胞和肿瘤细胞等主动分泌产生，exosomes广泛分布于外周血、尿液、唾液、乳汁、腹水、羊水等体液中。

    exosomes携带大量特异性的蛋白质（如细胞因子、生长因子）以及功能性的mRNAs、miRNAs等生物活性物质，在体内参与细胞通讯、细胞迁移、促血管新生和抗肿瘤免疫等生理过程，与多种疾病的发生和进程密切相关。由于外泌体的特殊结构和功能，使得它具有潜在的应用价值。

exosome提取方法比较 ——如何获得你所想要的exosomes？一方面可以作为诊断多种疾病的生物指标，

exosome提取方法比较 ——如何获得你所想要的exosomes？另一方面也可以作为治疗手段，未来有可能作为药物的天然载体用于临床治疗。

    外泌体的分离纯化一直是科研工作者关注的问题，exosome提取方法比较 ——如何获得你所想要的exosomes？获得高纯度的外泌体对后续的研究至关重要。据了解，目前人们多采用超速离心、免疫磁珠、超滤、沉淀或试剂盒等方法实现外泌体的提取分离：

1、超速离心法（差速离心）

超离法是最常用的外泌体纯化手段，采用低速离心、高速离心交替进行（如图所示），可分离到大小相近的囊泡颗粒。超离法因操作简单，获得的囊泡数量较多而广受欢迎，但过程比较费时，且回收率不稳定（可能与转子类型有关），纯度也受到质疑；此外，重复离心操作还有可能对囊泡造成损害，从而降低其质量。

2、密度梯度离心

在超速离心力作用下，使蔗糖溶液形成从低到高连续分布的密度阶层，是一种区带分离法。通过密度梯度离心，样品中的外泌体将在1.13-1.19g/ml的密度范围富集。此法获得的外泌体纯度较高，但步骤繁琐，耗时。

3、超滤离心

由于外泌体是一个大小约几十纳米的囊状小体，大于一般蛋白质，利用不同截留相对分子质量(MWCO)的超滤膜对样品进行选择性分离，便可获得外泌体。超滤离心法简单高效，且不影响外泌体的生物活性，是提取细胞外泌体的一种新方法。

4、磁珠免疫法

外泌体表面有其特异性标记物（如CD63、CD9蛋白），用包被抗标记物抗体的磁珠与外泌体囊泡孵育后结合，即可将外泌体吸附并分离出来。磁珠法具有特异性高、操作简便、不影响外泌体形态完整等优点，但是效率低，外泌体生物活性易受pH和盐浓度影响，不利于下游实验，难以广泛普及。

5、PEG-base沉淀法

   一篇来自《scientific reports》，IF=5.578的文献，文章标题是ExtraPEG: A Polyethylene Glycol-Based Method for Enrichment of ExtracellularVesicles（ExtraPEG:用聚乙二醇法浓缩胞外囊泡）。用聚乙二醇来纯化外泌体，首先，得确定加入的聚乙二醇的最适浓度是多少，因此作者分别选取5%-12%这8个浓度来探究哪个浓度更适合用于提取外泌体。利用PEG沉淀外泌体存在不少问题：比如纯度和回收率低，杂蛋白较多（假阳性），颗粒大小不均一，产生难以去除的聚合物，机械力或者吐温-20等化学添加物将会破坏外泌体等，因此发表文章时易受质疑。

6、试剂盒提取

    近几年来，市场上已出现各种商业化的外泌体提取试剂盒，有的通过特殊设计的过滤器过滤掉杂质成分，有的则采用空间排阻色谱法(SEC)进行分离纯化，也有的则利用化合物沉淀将法外泌体沉淀。exosome提取方法比较 ——如何获得你所想要的exosomes？这些试剂盒不需要特殊设备，随着产品不断更新换代，提取效率和纯化效果逐渐提高，因而逐渐取代超速离心法并推广开来。exosome提取方法比较 ——如何获得你所想要的exosomes？有些试剂盒操作简便，不用超速离心，同时可获得高纯度和高回收率的外泌体——如101bio开发的系列试剂盒，101bio开发的系列试剂盒分别针对尿液、血液、细胞上清液等多种样品进行提取，并可进一步从外泌体中获得想要的蛋白质或者RNA分子，方便快速，因而受到大多数人的欢迎，并发表了不少高质量的文章！

   exosome提取方法比较 ——如何获得你所想要的exosomes？市场上各类产品纯化效果良莠不齐，目前仍没有绝对的方法或试剂盒能满足所有要求，从各类样品中分离到理想的外泌体。

参考文献：

卢婉, 杨人强, 王伶. 外泌体的研究进展[J]. 生命的化学, 2013, 04期(04).

Taixue An et al. Exosomes serve as tumour markers for personalized diagnostics owing to their important role in cancer metastasis[J]. Journal of Extracellular Vesicles 2015, 4: 27522.

- http://dx.doi.org/10.3402/jev.v4.27522.

Richard J. Lobb et al. Optimized exosome isolation protocol for cell culture supernatant and human plasma[J]. Journal of Extracellular Vesicles 2015, 4: 27031.

- http://dx.doi.org/10.3402/jev.v4.27031.

 胡国文,李青,牛鑫等. 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.

Zhang et al. Exosomes in cancer: small particle, big player[J]. Journal of Hematology & Oncology, 2015, 8:83. doi:10.1186/s13045-015-0181-x.

 Korbelik M et al. Ceramide and Sphingosine-1-Phosphate/Sphingosine act as Photodynamic Therapy-Elicited Damage-Associated Molecular Patterns: Release from Cells and Impact on Tumor-Associated Macrophages. Janal Bioanal Tech. S1:009. doi: 10.4172/2155-9872.S1-009.

Chen L., Chen R., Brigstock D. MicroRNA profiling of circulating exosomes during experimental liver fibrosis.Hepatology. AASLD abstract, 2014 Oct. (Presidential Honored Distinct Abstract). 

PureExo? Exosome Isolation kit (for cell culture media)     Cat.#: P100  Link

Citation 1:

Osteikoetxea X, Németh A, Sódar BW, Vukman KV, Buzás EI. Extracellular vesicles in cardiovascular diseases, are they Jedi or Sith? 
J Physiol. 2016 Feb 13. doi: 10.1113/JP271336    

Citation 2:

Korbelik M, Banáth J, Zhang W, Wong F, Bielawski J, Separovic D. Ceramide and Sphingosine-1-Phosphate/Sphingosine act as Photodynamic Therapy-Elicited Damage-Associated Molecular Patterns: Release from Cells and Impact on Tumor-Associated Macrophages. J Anal Bioanal Tech. S1:009. doi: 10.4172/2155-9872.S1-009 (2014)  

Citation 3:

Salama A, Fichou N, Allard M, Dubreil L, De Beaurepaire L, Viel A, Jégou D, B?sch S, Bach JM.MicroRNA-29b modulates innate and antigen-specific immune responses in mouse models of autoimmunity. PLoS One. 2014 Sep 9;9(9):e106153. doi: 10.1371/journal.pone.0106153.  (Sold through our European distributor - Gentaur.)

Citation 4:

Mar?dziak M, Marycz K, Lewandowski D, Siudzińska A, ?mieszek A. Static magnetic field enhances synthesis and secretion of membrane-derived microvesicles (MVs) rich in VEGF and BMP-2 in equine adipose-derived stromal cells (EqASCs)-a new approach in veterinary regenerative medicine. In Vitro Cell Dev Biol Anim. 2015 Mar;51(3):230-40. doi: 10.1007/s11626-014-9828-0.  

Citation 5:

Tomoyo Kawakubo-Yasukochi, Masahiko Morioka, Yoshikazu Hayashi, Takuya Nishinakagawa, Mai Hazekawa, Shintaro Kawano, Seiji Nakamura, Manabu Nakashima    The SQUU-B cell line spreads its metastatic properties to nonmetastatic clone SQUU-A from the same patient through exosomes. J of Oral Biosciences   2015/09

PureExo? citation from Japan distributor Cosmo indicates interesting exosome function: "exosome derived from highly metastatic SQUU-B cells conferred metastatic ability to nonmetastatic SQUU-A cells."

Citation 6:

William G. Whitford    Exosomes: A Major Phenomenon Flying Under the Radar cellculturedish.com  2015/09

Citation 7:

Bonafede R, Scambi I, Peroni D, Potrich V, Boschi F, Benati D, Bonetti B, Mariotti R.    Exosome derived from murine adipose-derived stromal cells: Neuroprotective effect on in vitro model of amyotrophic lateral sclerosis. Exp Cell Res.   2016 Jan 1;340(1):150-8. doi: 10.1016/j.yexcr.2015.12.009. Epub 2015 Dec 18.

Citation 8:

Zhou Y, Fang L, Yu Y, Niu J, Jiang L, Cao H, Sun Q, Zen K, Dai C, Yang J.    Erythropoietin protects the tubular basement membrane by promoting the bone marrow to release extracellular vesicles containing tPA-targeting miR-144. Am J Physiol Renal Physiol.   2016 Jan 1;310(1):F27-40. doi: 10.1152/ajprenal.00303.2015. Epub 2015 Oct 14.

Citation 9:

Campbell CR, Berman AE, Weintraub NL, Tang YL.    Electrical stimulation to optimize cardioprotective exosomes from cardiac stem cells. Med Hypotheses.   2016 Mar;88:6-9. doi: 10.1016/j.mehy.2015.12.022. Epub 2016 Jan 11.

Citation 10:

Zhu D, Liu Z, Wang F. Effect of exosome extracted from 3T3 on proliferation of mouse colorectal cancer cells CT26. Chin J of Exp Surg. 2015 July;32(7):1634-36

Citation 11:

Rager T, Olson J, Zhou Y, Wang Y, Besner G. Exosomes Secreted from Bone Marrow-Derived Mesenchymal Stem Cells Protect the Intestines from Experimental Necrotizing Enterocolitis. J Pediatr Surg. 2016 March;S0022-3468(16)00149-4

Citation 12:

Li X, Liu L, Yang J, Yu Y, Chai J, Wang L, Ma L, Yin H. Exosome Derived From Human Umbilical Cord Mesenchymal Stem Cell Mediates MiR-181c Attenuating Burn-induced Excessive Inflammation.EBioMedicine. 2016, http://dx.doi.org/10.1016/j.ebiom.2016.04.030

Citation 13:

Masahiko Moriokaa, Tomoyo Kawakubo-Yasukochia, Yoshikazu Hayashi, Mai Hazekawa, Takuya Nishinakagawa, Kazuhiko Ono, Shintaro Kawano, Seiji Nakamura, Manabu Nakashima. Exosomes from oral squamous carcinoma cell lines, SQUU-A and SQUU-B, define the tropism of lymphatic dissemination. J of Oral Biosciences. 2016, http://dx.doi.org/10.1016/j.job.2016.05.001i

PureExo? Exosome Isolation kit (for serum or plasma)     Cat.#: P101  Link

Citation 1:

Chen L, Chen R, Kemper S, Charrier A, Brigstock DR. Suppression of fibrogenic signaling in hepatic stellate cells by Twist1-dependent microRNA-214 expression: Role of exosomes in horizontal transfer of Twist1. Am J Physiol Gastrointest Liver Physiol. 2015 Sep 15;309(6):G491-9. doi: 10.1152/ajpgi.00140.2015. Epub 2015 Jul 30.  

This paper is awarded TOP 10 best original research papers published by the American Physiological Society (APS Select). Link

(APSselect is a collection of the very best original research papers published by the American Physiological Society. Each month, the most outstanding recently published papers from our ten research journals are selected and made available through this multi-journal website.)

Citation 2:

Osteikoetxea X, Németh A, Sódar BW, Vukman KV, Buzás EI. Extracellular vesicles in cardiovascular diseases, are they Jedi or Sith? 
J Physiol. 2016 Feb 13. doi: 10.1113/JP271336    

Citation 3:

William G. Whitford    Exosomes: A Major Phenomenon Flying Under the Radar cellculturedish.com  2015/09

Citation 4:

Chen L., Chen R., Brigstock D. MicroRNA profiling of circulating exosomes during experimental liver fibrosis. Hepatology. AASLD abstract, 2014 Oct. (Presidential Honored Distinct Abstract).  

Exosomal RNA and Protein Extraction Kit     Cat.#: P200  Link

Citation 1:

Li X, Liu L, Yang J, Yu Y, Chai J, Wang L, Ma L, Yin H. Exosome Derived From Human Umbilical Cord Mesenchymal Stem Cell Mediates MiR-181c Attenuating Burn-induced Excessive Inflammation.EBioMedicine. 2016, http://dx.doi.org/10.1016/j.ebiom.2016.04.030

ExoFectin? sRNA-into-Exosome Kit (Chemical)     Cat.#: P401  Link

Citation 1:

Salama A, Fichou N, Allard M, Dubreil L, De Beaurepaire L, Viel A, Jégou D, B?sch S, Bach JM.MicroRNA-29b modulates innate and antigen-specific immune responses in mouse models of autoimmunity. PLoS One. 2014 Sep 9;9(9):e106153. doi: 10.1371/journal.pone.0106153.  (Sold through our European distributor Gentaur.)

ExoFectin? sRNA-into-Exosome Kit (Electro)     Cat.#: P400  Link

Citation 1:

MARTINEZ N., SAITTA S., PATACQ C. Utilisation d’exosomes comme bithérapie contre le cancer du colon . Projet de recherche 2013-2014.

Plasma Membrane Protein Extraction Kit (mammalian cells or tissues)     Cat.#: P503  Link

Citation 1:

Shibata T, Takahashi K, Matsubara Y, Inuzuka E, Nakashima F, Takahashi N, Kozai D, Mori Y, Uchida K. Identification of a prostaglandin D2 metabolite as a neuritogenesis enhancer targeting the TRPV1 ion channel. Sci Rep. 2016 Feb 16;6:21261. doi: 10.1038/srep21261.    

8-min. Cytoplasmic & Nuclear Protein Extraction Kit (cells & tissues)     Cat.#: P504  Link

Citation 1:

Frismantiene A, Dasen B, Pfaff D, Erne P, Resink TJ, Philippova M. T-cadherin promotes vascular smooth muscle cell dedifferentiation via a GSK3β-inactivation dependent mechanism. Cell Signal. 2016 Feb 22;28(5):516-530. doi: 10.1016/j.cellsig.2016.02.014.    

Citation 2:

Matsumura K, Nakazawa T, Nagayasu K, Gotoda-Nishimura N, Kasai A, Hayata-Takano A, Shintani N, Yamamori H, Yasuda Y, Hashimoto R, Hashimoto H. De novo POGZ mutations in sporadic autism disrupt the DNA-binding activity of POGZ. J Mol Psychiatry. 2016 Apr 21;4:1. doi: 10.1186/s40303-016-0016-x. eCollection 2016.

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3D Cell Culture Gel - Col-Tgel     Cat.#: P720  Link

Citation 1:

Kuwahara, Kenrick, Zhi Yang, Ginger C. Slack, Marcel E. Nimni and Bo Han. Cell delivery using an injectable and adhesive transglutaminase–gelatin gel. Tissue Engineering Part C: Methods 16, no. 4 (2009): 609-618.

Citation 2:

Kuwahara, Kenrick, Josephine Y. Fang, Zhi Yang and Bo Han. Enzymatic crosslinking and degradation of gelatin as a switch for bone morphogenetic protein-2 activity. Tissue Engineering Part A 17, no. 23-24 (2011): 2955-2964.

Citation 3:

Fang, Josephine, Zhi Yang, ShihJye Tan, Charisse Tayag, Marcel E. Nimni, Mark Urata and Bo Han.Injectable gel graft for bone defect repair. Regenerative medicine 9, no. 1 (2014): 41-51.

Citation 4:

Tan, ShihJye, Josephine Y. Fang, Zhi Yang, Marcel E. Nimni and Bo Han. The synergetic effect of hydrogel stiffness and growth factor on osteogenic differentiation. Biomaterials 35, no. 20 (2014): 5294-5306.

Citation 5:

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