方法文章

Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

DOI：

10.3791/63614

⸱

2022年5月13日

 ,  , 
1Department of Biology, School of Science, Penn State Erie, The Behrend College, 2Magee Womens Research Institute—Allied Member

本文内容

摘要

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The protocol here demonstrates that extracellular vesicles can be adequately separated from conditioned cell culture media using size exclusion chromatography.

摘要

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Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain proteins, nucleic acids, and lipids that are reflective of the parent cell from which they are derived. Proper separation of EVs from other components in a sample allows for characterization of their associated cargo and lends insight into their potential as intercellular communicators and non-invasive biomarkers for numerous diseases. In the current study, oligodendrocyte derived EVs were isolated from cell culture media using a combination of state-of-the-art techniques, including ultrafiltration and size exclusion chromatography (SEC) to separate EVs from other extracellular proteins and protein complexes. Using commercially available SEC columns, EVs were separated from extracellular proteins released from human oligodendroglioma cells under both control and endoplasmic reticulum (ER) stress conditions. The canonical EV markers CD9, CD63, and CD81 were observed in fractions 1-4, but not in fractions 5-8. GM130, a protein of the Golgi apparatus, and calnexin, an integral protein of the ER, were used as negative EV markers, and were not observed in any fraction. Further, when pooling and concentrating fractions 1-4 as the EV fraction, and fractions 5-8 as the protein fraction, expression of CD63, CD81, and CD9 in the EV fraction was observed. The expression of GM130 or calnexin was not observed in either of the fraction types. The pooled fractions from both control and ER stress conditions were visualized with transmission electron microscopy and vesicles were observed in the EV fractions, but not in the protein fractions. Particles in the EV and protein fractions from both conditions were also quantified with nanoparticle tracking analysis. Together, these data demonstrate that SEC is an effective method for separating EVs from conditioned cell culture media.

引言

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The explosion of interest in studying extracellular vesicles (EVs) has been accompanied by major advancements in the technologies and techniques used to separate and study these nano-sized, heterogeneous particles. In the time since their discovery nearly four decades ago1,2, these small membranous structures have been found to contain bioactive lipids, nucleic acids, and proteins, and play major roles in intercellular communication3,4. EVs are released from all cell types and are therefore present in all biological fluids, including blood plasma and s....

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方案

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1. Preparation of buffers and reagents

NOTE: Make cell culture reagents in cell culture hood to maintain sterility.

  1. Preparation of cell culture reagents
    1. Prepare normal high glucose DMEM by adding 50 mL of FBS and 5 mL of penicillin-streptococcus (Pen-Strep) into 500 mL of high glucose DMEM and store at 4 °C. Use this media for culturing and expanding cells.
    2. Prepare exosome-depleted high glucose DMEM by adding 50 mL of exosome-depleted FBS and 5 mL of Pen-Strep into 500 mL of high glucose DMEM and store at 4 °C. Use this media for cell treatments prior to EV isolation.
      3. ....

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结果

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Western blotting reveals adequate separation of EVs from CCM
To evaluate the effectiveness of SEC for separating EVs from cell culture media, a western blot was run using each individual fraction from the control samples to probe expression of the three canonical EV markers, CD9, CD63 and CD81, as well as GM130 and calnexin18, which were used as negative controls (Figure 3). Albumin expression18 was also probed to ensure th.......

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讨论

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SEC is a user-friendly method for adequately separating EVs from conditioned CCM. In order to specifically isolate cell derived EVs, careful consideration of the type of CCM and its supplements must be taken into account. Many cell culture medias need to be supplemented with FBS, which contains EVs derived from the animal in which the serum was harvested. These serum EVs may saturate and mask any signal produced by EVs derived from cells in culture26. Therefore, when performing experiments, EV-dep.......

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披露

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The authors declare no conflicts of interest.

致谢

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The authors would like to thank Penn State Behrend and the Hamot Health Foundation for funding, as well as the Penn State Microscopy Facility in University Park, PA.

....

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材料

本文使用的材料清单
姓名公司目录编号评论
2-巯基乙醇VWR97064-588
4X Laemmli 样品缓冲液BioRad1610747
Amicon Ultra-15 离心过滤装置，Ultracel，3 KDa，15 mLSigma-AldrichUFC9003083 kDa 截留
Amicon Ultra-2 离心过滤装置，带 Ultracel-3 膜Sigma-AldrichUFC2003243 kDa 截留
铵过硫酸盐Sigma-AldrichA3678-100G
抗兔 IgG， HRP 联动抗体Cell Signaling Technology7074V1：1000 稀释
抗钙联蛋白抗体Abcam ab225951：500 稀释
CD9 小鼠单克隆抗体BioLegend3121021：500 稀释
GM130 抗体 [EP892Y] - 顺式高尔基体标志物Abcamab526491：500 稀释
抗小鼠 IgG、HRP 偶联抗体Cell Signaling Technology7076V1：1000 稀释
自动馏分收集器Izon Science
BCA 检测试剂盒Bio-Rad
CCD 相机Gatan Orius SC200
Cd63 小鼠抗BD5560191：1000 稀释
CD81 抗体Santa Cruz Biotechnologysc-239621：1000 稀释
Cellstar 过滤器 盖 细胞培养瓶Greiner Bio-One660175
ChemiDoc MP成像仪BioRad
Clarity Western ECL 底物BioRad1705061
脱氧胆酸盐Sigma-AldrichD6750-10G
二硫苏糖醇Sigma3483-12-3
DMEM/含 L-谷氨酰胺的高葡萄糖;不含Cytiva钠 SH300022。
胎牛血清 优质级VWR97068-085
胎牛血清，外泌体耗尽Thermo ScientificA2720801
甘氨酸BioRad1610718
超值脱脂奶粉AmazonB076NRD2TZ
HOG 人少突胶质细胞瘤细胞系Sigma-AldrichSCC163
Izon Science Usa Ltd qev 尺寸排阻柱 5 件装Izon Science
甲醇 >99.8% ACSVWRBDH1135-4LP
Mini-PROTEAN 玻璃板BioRad1653310，带 0.75 mm 垫片
Mini-PROTEAN 短板BioRad1653308
NP-40Sigma-Aldrich492016
青霉素-链霉素，溶液Sigma-AldrichP4458-100mL
磷酸盐缓冲盐水 PBSFisher ScientificBP66150
Pierce BCA 蛋白检测试剂盒和试剂Thermo Fisher Scientific23227
Pierce PVDF 转印膜Thermo Scientific88518
Pierce Western 印迹滤纸Thermo科学产品84783
聚氧乙烯-20 （吐温 20），500 mL生物碱性TB0560
蛋白酶/磷酸酶抑制剂混合物 （100X）Cell Signaling Technology5872S
重组抗 TSG101 抗体 [EPR7130（B）]ABCamab1250111：1000 稀释氢
氧化铑Sigma-AldrichSX0603
叠氮化钠Fisher ScientificBP922I-500
氯化钠Sigma-AldrichS9888-500G
十二烷基硫酸钠，≥99.0% （GC），无尘颗粒Sigma-Aldrich75746-1KG
四甲基乙二胺Sigma-AldrichT9281-25ML
TGX 免染 FastCast 丙烯酰胺套件，10%BioRad1610183
透射电子显微镜FEI Tecnai 12 Biotwin
TrisBioRad1610716
胰蛋白酶 0.25% 蛋白酶与猪胰蛋白酶、HBSS、EDTA;不含钙、镁CytivaSH30042.01
衣霉素Tocris3516
Zeta View 软件AnalytikNTA 软件
量 人 度 FS

参考文献

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