PDF(Pubmed)
Abstract：
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.
摘要：
细胞外囊泡（EV）具有为骨骼肌（SM）生理学和病理生理学提供新见解的潜力。然而,目前的分离方案通常不能消除共同分离的成分,如脂蛋白和RNA结合蛋白,这些成分可能混淆结果并阻碍下游临床翻译.在这项研究中,我们验证了EV分离方案，该方案将尺寸排阻色谱（SEC）与超滤（UF）相结合，以增加样品通量，可扩展性和纯度，同时首次分析了UF柱选择和分数窗口对EV回收率的影响。C2C12肌管条件培养基使用Amicon®Ultra15或Vivaspin®20100KDaUF柱预浓缩，并通过SEC(IZON，qEV70nm）。单独分析所得的30个馏分,以确定EV回收的最佳馏分窗口。EV标记物TSG101可以从级分5至14检测到,而CD9和膜联蛋白A2仅达到级分6。对于两种方案,从级分6开始检测ApoA1+脂蛋白共分离物。引人注目的是，Amicon和VivaspinUF浓缩方案导致EV标记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隔离导致颗粒净损失，但增加了样品纯度的测量，对EV标记蛋白的存在只有微不足道的影响。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(分数1-5)和总EV丰度(分数2-10)的EV分离.我们提供了证据来证明UF柱的选择会影响所得EV制剂的组成，并且在SM中的EV分离研究中应用时需要考虑。所得到的方案在分离用于一系列治疗和诊断研究中的应用的高纯度EV制剂方面将是有价值的。
