Defining the influence of size-exclusion chromatography fraction window and ultrafiltration column choice on extracellular vesicle recovery in a skeletal muscle model
Defining the influence of size-exclusion chromatography fraction window and ultrafiltration column choice on extracellular vesicle recovery in a skeletal muscle model
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定义尺寸排阻色谱分数窗口和超滤柱选择对骨骼肌模型中细胞外囊泡回收的影响
DOI：
10.1002/jex2.85
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Fernández-Rhodes M
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作者：
Fernández-Rhodes M
细胞外囊泡（EV）有可能为骨骼肌（SM）生理学和病理生理学提供新的见解。然而，目前的分离方案通常不能消除共分离的组分，如脂蛋白和RNA结合蛋白，这些组分可能会混淆结果并阻碍下游临床转化。在这项研究中，我们验证了一种EV分离方案，该方案将尺寸排阻色谱（SEC）与超滤（UF）相结合，以提高样品通量，可扩展性和纯度，同时首次分析了UF柱选择和馏分窗口对EV回收率的影响。使用Amicon®Ultra 15或Vivaspin®20 100 KDa UF柱预浓缩C2 C12肌管条件培养基，并通过SEC（IZON，qEV 70 nm）处理。单独分析得到的30个级分，以确定EV回收的最佳级分窗口。EV标志物TSG 101可以从级分5至14中检测到，而CD 9和膜联蛋白A2仅直到级分6。对于两种方案，从组分6开始检测ApoA 1+脂蛋白共分离株。引人注目的是，Amicon和Vivaspin UF浓缩方案导致EV标志物谱和纯度的定性和定量变化。通过减少SEC分数窗口消除脂蛋白共分离导致颗粒的净损失，但增加了样品纯度的测量值，并且对EV标志物蛋白的存在仅具有可忽略的影响。总之，我们的研究开发了一种有效的UF+SEC方案，用于基于样品纯度（级分1-5）和总EV丰度（级分2-10）分离EV。我们提供的证据表明，UF柱的选择可以影响所得EV制剂的组成，并需要考虑应用于SM EV分离研究时。由此产生的协议将是有价值的分离高纯度EV制剂的应用范围内的治疗和诊断研究。
Extracellular vesicles (EVs) have the potential to provide new insights into skeletal muscle (SM) physiology and pathophysiology. However, current isolation protocols often do not eliminate co‐isolated components such as lipoproteins and RNA binding proteins that could confound outcomes and hinder downstream clinical translation. In this study, we validated an EV isolation protocol that combined size‐exclusion chromatography (SEC) with ultrafiltration (UF) to increase sample throughput, scalability and purity, while providing the very first analysis of the effects of UF column choice and fraction window on EV recovery. C2C12 myotube conditioned medium was pre‐concentrated using either Amicon®Ultra 15 or Vivaspin®20 100 KDa UF columns and processed by SEC (IZON, qEV 70 nm). The resulting thirty fractions obtained were individually analysed to identify an optimal fraction window for EV recovery. The EV marker TSG101 could be detected from fractions 5 to 14, while CD9 and Annexin A2 only up to fraction 6. ApoA1+lipoprotein co‐isolates were detected from fraction 6 onwards for both protocols. Strikingly, Amicon and Vivaspin UF concentration protocols led to qualitative and quantitative variations in EV marker profiles and purity. Eliminating lipoprotein co‐isolation by reducing the SEC fraction window resulted in a net loss of particles, but increased measures of sample purity and had only a negligible impact on the presence of EV marker proteins. In conclusion, our study developed an effective UF+SEC protocol for the isolation of EVs based on sample purity (fractions 1–5) and total EV abundance (fractions 2–10). We provide evidence to demonstrate that the choice of UF column can affect the composition of the resulting EV preparation and needs to be considered when being applied in EV isolation studies in SM. The resulting protocols will be valuable in isolating highly pure EV preparations for applications in a range of therapeutic and diagnostic studies.