一种从人间充质干细胞中分离小细胞外囊泡的简单台式过滤方法
1Centre for Tissue Engineering and Regenerative Medicine,Universiti Kebangsaan Malaysia Medical Centre, 2My CytoHealth Sdn. Bhd., 3School of Biosciences, Faculty of Health and Medical Sciences,Taylor’s University, 4School of Pharmacy,Monash University Malaysia, 5Haematology Unit, Cancer Research Centre, Institute for Medical Research (IMR),National Institutes of Health (NIH), Ministry of Health Malaysia, 6School of Pharmacy, Faculty of Health & Medical Sciences,Taylor’s University, 7Centre for Drug Discovery and Molecular Pharmacology (CDDMP), Faculty of Health and Medical Sciences,Taylor’s University
Summary
该协议演示了如何使用简单的实验室规模台式设置分离人脐带衍生间充质干细胞的小细胞外囊泡（hUC-MSC-sEVs）。采用纳米颗粒示踪分析、BCA蛋白测定、蛋白质印迹和透射电子显微镜分别对分离的hUC-MSC-sEV的粒径分布、蛋白浓度、sEVs标志物和形貌进行了表征。
Abstract
基于超速离心的过程被认为是小细胞外囊泡（sEV）分离的常用方法。然而，这种分离方法的产量相对较低，并且这些方法在分离sEV亚型方面效率低下。本研究展示了一种简单的台式过滤方法，用于分离人脐带衍生的MSC小细胞外囊泡（hUC-MSC-sEVs），通过超滤成功地从hUC-MSC的条件培养基中分离出来。采用纳米颗粒示踪分析、BCA蛋白测定、蛋白质印迹和透射电镜分别对分离的hUC-MSC-sEVs的尺寸分布、蛋白浓度、外泌体标志物（CD9、CD81、TSG101）和形貌进行了表征。分离出的hUC-MSC-sEVs的尺寸为30-200 nm，颗粒浓度为7.75 ×10 10 颗粒/mL，蛋白质浓度为80 μg/mL。观察到外泌体标志物CD9，CD81和TSG101的阳性条带。本研究表明，hUC-MSC-sEVs已成功从hUC-MSCs条件培养基中分离出来，表征表明分离出的产物符合2018年细胞外囊泡研究最小信息（MISEV 2018）提到的标准。
Introduction
根据MISEV 2018，sEV是非复制的脂质双层颗粒，不存在功能核，尺寸为30-200 nm1。MSC 衍生的 sEV 含有重要的信号分子，在组织再生中起重要作用，例如 microRNA、细胞因子或蛋白质。它们日益成为再生医学和无细胞疗法的研究“热点”。许多研究表明，间充质干细胞衍生的sEV在治疗不同疾病方面与间充质干细胞一样有效，例如免疫调节2，3，4，5，增强成骨6，糖尿病7，8或血管再生9，10。随着早期试验的进展，与MSCs-EVs的临床转化相关的三个主要关键问题得到了强调：EVs的产量，EVs的纯度（不含细胞碎片和其他生物污染物，如蛋白质和细胞因子），以及分离后EV磷脂双层膜的完整性。
已经开发了各种方法来分离sEV，利用sEV的密度，形状，大小和表面蛋白11。sEV分离中最常见的两种方法是基于超速离心和基于超滤的技术。
基于超速离心的方法被认为是sEV分离的金标准方法。通常采用的两种类型的超速离心技术是差分超速离心和密度梯度超速离心。然而，超速离心方法通常会导致低产量，并且需要昂贵的高速超速离心设备（100，000-200，000 × g）11。此外，单独的超速离心技术在分离EV亚型（sEV和大型EV）方面效率低下，导致沉积层不纯11。最后，密度梯度超速离心也可能非常耗时，并且需要额外的预防措施，例如添加蔗糖缓冲液以抑制加速和减速步骤12期间的梯度损伤。因此，超速离心通常会导致相对较低的产率，并且无法区分不同EV群体13，这限制了其在大规模EV制备中的应用11。
EV隔离的第二种方法是通过超滤，它基于尺寸过滤。与超速离心相比，超滤相对省时且具有成本效益，因为它不涉及昂贵的设备或较长的处理时间14。因此，超滤似乎是一种比上述两种超速离心方法更有效的分离技术。分离的产品可以根据孔径和更高的产量更具体15。然而，在过滤过程中产生的额外力可能导致EV变形或喷发16。
本文提出了一种经济高效的台式方案，用于分离MSC衍生的sEV用于下游分析和治疗目的。本文描述的方法将简单的过滤方法与台式离心相结合，从hUC-MSC中分离出高产量和高质量的EV，用于下游分析，包括粒度分析，生物标志物测定和电子显微镜成像。
Protocol
注意：有关此协议中使用的所有材料、设备和软件的详细信息，请参阅 材料表 。 1. 人脐带间充质干细胞及培养 在DMEM中以5×103 / cm2 的接种密度培养hUC-MSC，并补充有8%的人血小板裂解物和1%笔链球菌。将细胞在37°C的5%CO2中孵育。每3天更换一次细胞培养基，以确保细胞正常生长。 将细胞扩增至T175烧瓶中的传代5?…
Representative Results
图2 显示hUC-MSC-sEV在53 nm处具有粒径模式，而其他显著的粒径峰分别为96和115 nm。NTA测量的hUC-MSC-sEVs浓度为7.75×1010 个颗粒/mL。用BCA测定法测量的hUC-MSC-sEVs的蛋白质浓度约为80μg/ mL。 在蛋白质印迹分析中，hUC-MSC-sEVs的外泌体标志物CD9、CD81和TSG101呈阳性条带，但GRP94呈阴性（图3）。GRP94是一种内质网标记物，通常用作sEV的阴性?…
Discussion
EV是间充质干细胞分泌组的重要子集之一，在正常和病理过程中起着至关重要的作用。然而，在过去十年中，尺寸范围在30至200nm之间的sEV已成为无细胞治疗的潜在工具。开发了各种技术来从间充质干细胞中分离sEV。然而，差示超速离心、超滤、基于聚合物的沉淀、免疫亲和捕获和基于微流体的沉淀具有不同的优缺点17。这些分离技术都无法实现高回收率和特异性。因此，研究人?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
该视频的发布得到了My CytoHealth Sdn. Bhd的支持。
Materials
| 40% acrylamide | Nacalai Tesque | 06121-95 | Western blot |
| 95% ethanol | Nacalai Tesque | 14710-25 | Disinfectants |
| Absolute Methanol | Chemiz | 45081 | To activate PVDF membrane (Western blot) |
| Accutase | STEMCELL Technologies | 7920 | Cell dissociation enzyme |
| ammonium persulfate | Chemiz | 14475 | catalyse the gel polymerisation (Gel electrophoresis |
| anti-CD 81 (B-11) | Santa Cruz Biotechnology | sc-166029 | Antibody for sEVs marker |
| anti-TSG 101 (C-2) | Santa Cruz Biotechnology | sc-7964 | Antibody for sEVs marker |
| Bovine serum albumin | Nacalai Tesque | 00653-31 | PVDF membrane blocking |
| bromophenol blue | Nacalai Tesque | 05808-61 | electrophoretic color marker |
| Centricon Plus-70 (100 kDa NMWL) | Millipore | UFC710008 | sEVs isolation |
| ChemiLumi One L | Nacalai Tesque | 7880 | chemiluminescence detection reagent |
| CryoStor Freezing Media | Sigma-Aldrich | C3124-100ML | Cell cryopreserve |
| Dulbecco’s modified Eagle’s medium | Nacalai Tesque | 08458-45 | Cell culture media |
| ExcelBand Enhanced 3-color High Range Protein Marker | SMOBIO | PM2610 | Protein molecular weight markers |
| Extra thick blotting paper | ATTO | buffer reservior (Western blot) | |
| Glycerol | Merck | G5516 | Chemicals for western blot |
| Glycine | 1st Base | BIO-2085-500g | Chemicals for buffer (Western Blot) |
| horseradish peroxidase-conjugated mouse IgG kappa binding protein (m-IgGκBP-HRP) | Santa Cruz Biotechnology | sc-516102 | Secondary antibody (Western Blot) |
| Human Wharton’s Jelly derived Mesenchymal Stem Cells (MSCs) | Centre for Tissue Engineering and Regenerative Medicine, Faculty of Medicine, The National University Malaysia | ||
| mouse antibodies anti-CD 9 (C-4) | Santa Cruz Biotechnology | sc-13118 | Antibody for sEVs marker |
| Nanosight NS300 equipped with a CMOS camera, a 20 × objective lens, a blue laser module (488 nm), and NTA software v3.2 | Malvern Panalytical, UK | ||
| paraformaldehyde | Nacalai Tesque | 02890-45 | Sample Fixation during TEM |
| penicillin–streptomycin | Nacalai Tesque | 26253-84 | Antibiotic for media |
| phenylmethylsulfonyl fluoride | Nacalai Tesque | 27327-81 | Inhibit proteases in the sEVs samples after adding lysis buffer |
| phosphate-buffered saline | Gibco | 10010023 | Washing, sample dilution |
| polyvinylidene fluoride (PVDF) membrane with 0.45 mm pore size |
ATTO | To hold protein during protein transfer (Western blot) | |
| protease inhibitor cocktail | Nacalai Tesque | 25955-11 | Inhibit proteases in the sEVs samples after adding lysis buffer |
| Protein Assay Bicinchoninate Kit | Nacalai Tesque | 06385-00 | Protein measurement |
| sample buffer solution with 2-ME | Nacalai Tesque | 30566-22 | Reducing agent for western blot |
| sodium chloride | Nacalai Tesque | 15266-64 | Chemicals for western blot |
| sodium dodecyl sulfate | Nacalai Tesque | 31606-62 | ionic surfactant during gel electrophoresis |
| Tecnai G2 F20 S-TWIN transmission electron microscope | FEI, USA | ||
| tetramethylethylenediamine | Nacalai Tesque | 33401-72 | chemicals to prepare gel |
| tris-base | 1st Base | BIO-1400-500g | Chemicals for buffer (Western Blot) |
| Tween 20 | GeneTex | GTX30962 | Chemicals for western blot |
| UVP (Ultra Vision Product) CCD imager | CCD imager for western blot signal detection |
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Citazione di questo articolo
Koh, B., Tan, K. L., Chan, H. H., Daniel Looi, Q. H., Lim, M. N., How, C. W., Law, J. X., Foo, J. B. A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells. J. Vis. Exp. (184), e64106, doi:10.3791/64106 (2022).