用于从条件细胞培养基中分离细胞外囊泡的体积排阻色谱
概要
这里的方案表明，可以使用尺寸排阻色谱从条件细胞培养基中充分分离细胞外囊泡。
Abstract
细胞外囊泡（EV）是纳米大小的脂膜结合结构，从所有细胞释放，存在于所有生物流体中，并含有蛋白质，核酸和脂质，这些蛋白质，核酸和脂质反映了它们来源的母细胞。将EV与样品中的其他成分适当分离可以表征其相关货物，并深入了解它们作为细胞间通讯剂和多种疾病的非侵入性生物标志物的潜力。在目前的研究中，使用最先进的技术（包括超滤和体积排阻色谱（SEC））从细胞培养基中分离出少突胶质细胞衍生的EV，以将EV与其他细胞外蛋白质和蛋白质复合物分离。使用市售的SEC色谱柱，在对照和内质网（ER）应激条件下，将EVs与人少突胶质瘤细胞释放的细胞外蛋白分离。在分数 1-4 中观察到典型的 EV 标记 CD9、CD63 和 CD81，但在分数 5-8 中未观察到。高尔基体的一种蛋白质GM130和ER的整合蛋白calnexin被用作负EV标志物，并且未在任何部分中观察到。此外，当汇集和浓缩级分 1-4 作为 EV 级分，级分 5-8 作为蛋白质级分时，观察到 EV 级分中 CD63、CD81 和 CD9 的表达。在两种馏分类型中均未观察到GM130或钙连接蛋白的表达。使用透射电子显微镜观察来自对照和ER应力条件的混合级分，并且在EV级分中观察到囊泡，但在蛋白质级分中未观察到囊泡。两种条件下的EV和蛋白质级分中的颗粒也通过纳米颗粒跟踪分析进行定量。这些数据共同表明，SEC是从条件细胞培养基中分离EV的有效方法。
Introduction
对研究细胞外囊泡（EVs）的兴趣激增，伴随着用于分离和研究这些纳米大小的异质颗粒的技术和技术的重大进步。自近四十年前发现以来1，2，这些小膜结构被发现含有生物活性脂质，核酸和蛋白质，并在细胞间通讯中起主要作用3，4。EV从所有细胞类型中释放出来，因此存在于所有生物体液中，包括血浆和血清，唾液和尿液。这些液体中的电动汽车有望作为各种疾病的非侵入性生物标志物，包括神经炎症和神经退行性疾病、癌症和自身免疫性疾病5，6，7。此外，体外机理研究可以通过细胞培养技术通过分离释放到培养基中的EV来进行3，8，9。
要了解 EV 在疾病病理生理学中的作用，与发现它们的液体充分分离至关重要。长期以来，EV分离的黄金标准一直是差示超速离心（dUC）10，但是已经出现了更复杂的技术来实现EV与其他细胞外成分的更好分离。其中一些技术包括密度梯度、不对称流场流分数 （A4F）、流式细胞术、免疫捕获、聚乙二醇沉淀和体积排阻色谱 （SEC）11，12，13。每种技术都有自己的一套优点和缺点;然而，SEC尤其被证明可以非常有效地将EV从生物体液和细胞培养上清液中分离出来8，14，15。SEC还有一个额外的好处，即相对简单和用户友好。
SEC是一种根据尺寸分离流体成分的方法。使用这种技术，使用树脂柱（内部制造或商业购买）对样品进行分馏。样品中的小颗粒被困在树脂内的珠子之间，而较大的颗粒能够更自由地通过树脂，从而在过程中更早地洗脱。由于 EV 的尺寸大于许多细胞外蛋白和蛋白质聚集体，因此 EV 比细胞外蛋白更快地通过色谱柱并洗脱更早的馏分14。
在该方法论文中，概述了在对照和内质网（ER）应激条件下使用SEC从细胞培养基（CCM）中分离人少突胶质细胞的EV。使用该协议，表明用该技术分离的EV存在于特定的部分中，这些部分可以汇集在一起并浓缩以进行下游表征，并且分离的EV来自细胞而不是外源，例如胎牛血清（FBS）用于补充CCM。蛋白质印迹法证明 EV 级分中存在典型的 EV 标志物 CD63、CD81 和 CD916、17、18、19，蛋白质级分中不存在这些标志物。使用透射电子显微镜（TEM），EV被可视化并显示预期的形态，并且仅在EV部分中观察到。在对照和ER应力条件下的EV和蛋白质级分中也计数颗粒，并且在EV样品中观察到直径为50-200nm的预期尺寸范围内的大量颗粒。这些数据共同支持了SEC是从细胞培养基中分离EV的高效方法的观点。
Protocol
1. 缓冲液和试剂的制备 注意：在细胞培养罩中制作细胞培养试剂以保持无菌。 细胞培养试剂的制备通过将50mL FBS和5mL青霉素链球菌（Pen-Strep）加入500 mL高葡萄糖DMEM中并储存在4°C来制备正常的高葡萄糖DMEM。 使用该培养基培养和扩增细胞。 通过将 50 mL 去除外泌体的 FBS 和 5 mL 笔链球菌加入 500 mL 高葡萄糖 DMEM 中制备外泌体去除的高葡萄糖 DMEM?…
Representative Results
蛋白质印迹显示EV与CCM充分分离为了评估SEC从细胞培养基中分离EV的有效性，使用对照样品中的每个单独级分进行蛋白质印迹，以检测三种典型EV标志物CD9，CD63和CD81以及用作阴性对照的GM130和calnexin18的表达（图3）。还研究了白蛋白表达18 ，以确保CCM中的细胞外蛋白可以与EV充分分离。在馏分 1-4 中观察到 CD9、CD63 和 CD81 的强…
Discussion
SEC是一种用户友好的方法，用于将EV与条件CCM充分分离。为了特异性分离细胞衍生的EV，必须仔细考虑CCM的类型及其补充剂。许多细胞培养基需要补充FBS，FBS含有来自收获血清的动物的EV。这些血清 EV 可能会饱和并掩盖源自培养细胞的 EV 产生的任何信号26。因此，在进行实验时，应尽可能使用EV耗尽的FBS，以确保发现的EV可以归因于细胞衍生的EV，而不是牛衍生的EV，这可以商业购?…
開示
The authors have nothing to disclose.
Acknowledgements
作者要感谢宾夕法尼亚州立大学贝伦德和哈莫特健康基金会的资助，以及宾夕法尼亚州大学公园的宾夕法尼亚州立大学显微镜设施。
Materials
| 2-Mercaptoethanol | VWR | 97064-588 | |
| 4X Laemmli Sample Buffer | BioRad | 1610747 | |
| Amicon Ultra-15 Centrifugal Filter Unit, Ultracel, 3 KDa, 15mL | Sigma-Aldrich | UFC900308 | 3 kDa cutoff |
| Amicon Ultra-2 Centrifugal Filter Unit with Ultracel-3 membrane | Sigma-Aldrich | UFC200324 | 3 kDa cutoff |
| Ammonium Persulfate | Sigma-Aldrich | A3678-100G | |
| Anti rabbit IgG, HRP linked Antibody | Cell Signaling Technology | 7074V | 1:1000 Dilution |
| Anti-Calnexin antibody | Abcam | ab22595 | 1:500 Dilution |
| Anti-CD9 Mouse Monoclonal Antibody | BioLegend | 312102 | 1:500 Dilution |
| Anti-GM130 antibody [EP892Y] – cis-Golgi Marker | Abcam | ab52649 | 1:500 Dilution |
| Anti-mouse IgG, HRP-linked Antibody | Cell Signaling Technology | 7076V | 1:1000 Dilution |
| Automatic Fraction Collector | Izon Science | ||
| BCA assay Kit | Bio-Rad | ||
| CCD camera | Gatan Orius SC200 | ||
| Cd63 Mouse anti Human | BD | 556019 | 1:1000 Dilution |
| CD81 Antibody | Santa Cruz Biotechnology | sc-23962 | 1:1000 Dilution |
| Cellstar Filter Cap Cell Culture Flasks | Greiner Bio-One | 660175 | |
| ChemiDoc MP Imager | BioRad | ||
| Clarity Western ECL Substrate | BioRad | 1705061 | |
| deoxycholate | Sigma-Aldrich | D6750-10G | |
| dithiothreitol | Sigma | 3483-12-3 | |
| DMEM/High glucose with L-glutamine; without sodium | Cytiva | SH300022.FS | |
| Fetal Bovine Serum Premium grade | VWR | 97068-085 | |
| Fetal Bovine Serum, exosome-depleted | Thermo Scientific | A2720801 | |
| Glycine | BioRad | 1610718 | |
| Great Value Nonfat Dry Milk | Amazon | B076NRD2TZ | |
| HOG Human Oligodendroglioma Cell Line | Sigma-Aldrich | SCC163 | |
| Izon Science Usa Ltd qev Size Exclusion Columns 5pk | Izon Science | ||
| Methanol >99.8% ACS | VWR | BDH1135-4LP | |
| Mini-PROTEAN Glass plates | BioRad | 1653310 | with 0.75mm spacers |
| Mini-PROTEAN Short plates | BioRad | 1653308 | |
| NP-40 | Sigma-Aldrich | 492016 | |
| Penicillin-Streptomycin,Solution | Sigma-Aldrich | P4458-100mL | |
| Phosphate Buffered Saline PBS | Fisher Scientific | BP66150 | |
| Pierce BCA Protein Assay Kits and Reagents | Thermo Fisher Scientific | 23227 | |
| Pierce PVDF Transfer Membranes | Thermo Scientific | 88518 | |
| Pierce Western Blotting Filter Paper | Thermo Scientific | 84783 | |
| Polyoxyethylene-20 (TWEEN 20), 500mL | Bio Basic | TB0560 | |
| Protease/phosphatase Inhibitor Cocktail (100X) | Cell Signaling Technology | 5872S | |
| Recombinant Anti-TSG101 antibody [EPR7130(B)] | ABCam | ab125011 | 1:1000 dilution |
| Slodium hydroxide | Sigma-Aldrich | SX0603 | |
| Sodium azide | Fisher Scientific | BP922I-500 | |
| Sodium Chloride | Sigma-Aldrich | S9888-500G | |
| Sodium dodecyl sulfate,≥99.0% (GC), dust-free pellets | Sigma-Aldrich | 75746-1KG | |
| Tetramethylethylenediamine | Sigma-Aldrich | T9281-25ML | |
| TGX Stain-Free FastCast Acrylamide Kit, 10% | BioRad | 1610183 | |
| Transmission Electron Microscope | FEI Tecnai 12 Biotwin | ||
| Tris | BioRad | 1610716 | |
| Trypsin 0.25% protease with porcine trypsin, HBSS, EDTA; without calcium, magnesium | Cytiva | SH30042.01 | |
| Tunicamycin | Tocris | 3516 | |
| Zeta View software | Analytik | NTA software |
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