Optimization of cell culture conditions for exosome isolation using mini-size exclusion chromatography (mini-SEC)
Optimization of cell culture conditions for exosome isolation using mini-size exclusion chromatography (mini-SEC)
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DOI：
10.1016/j.yexcr.2019.03.014
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发表时间：
2019-05-15
影响因子：
3.7
通讯作者：
Whiteside, Theresa L.
中科院分区：
文献类型：
--
作者：
Ludwig, Nils;Razzo, Beatrice M.;Whiteside, Theresa L.
细胞外囊泡（EVs）是一种重要的细胞间通讯系统，参与多种生理和病理生理过程。肿瘤来源的外泌体（TEX）是作为潜在癌症生物标志物特别感兴趣的EV的子集。肿瘤细胞系的上清液被广泛用作分子/遗传研究的纯TEX来源。为了优化这些研究的TEX分离和表征，我们评估了不同肿瘤细胞系的培养条件，并使用微型尺寸排阻色谱法（mini-SEC）进行TEX分离。每种肿瘤细胞系都显示出独特的培养要求，这些要求决定了TEX的回收率、纯度和总产率。通过改变培养基组成和接种细胞的数量，可以修改最佳TEX纯度和mini-SEC回收的培养条件。在优化条件下从mini-SEC级分#4回收的TEX是生物活性的，直径大小为30至150 nm，具有典型的囊泡形态并携带内吞标记物。可再现的外来体回收的最关键要求是重新接种肿瘤细胞，其数量经调整以反映每种肿瘤细胞系的最佳培养条件。这项研究提供了对细胞培养技术的见解，该技术可以通过各种人类或小鼠肿瘤细胞系优化外泌体生产，以通过mini-SEC分离。
Extracellular vesicles (EVs) are emerging as a major intercellular communication system engaged in a variety of physiological and pathophysiological processes. Tumor-derived exosomes (TEX) are a subset of EVs of special interest as potential cancer biomarkers. Supernatants of tumor cell lines are widely used as the source of pure TEX for molecular/genetic studies. To optimize TEX isolation and characterization for these studies, we evaluated culture conditions for different tumor cell lines and used mini size exclusion chromatography (mini-SEC) for TEX isolation. Each tumor cell line showed unique culture requirements that determined the recovery, purity and total yield of TEX. Culture conditions for optimal TEX purity and recovery by mini-SEC could be modified by altering the media composition and numbers of seeded cells. TEX recovered from mini-SEC fraction #4 under optimized conditions were biologically active, were sized from 30 to 150 nm in diameter, had a typical vesicular morphology and carried endocytic markers. The most critical requirement for reproducible exosome recovery was re-seeding of tumor cells in numbers adjusted to reflect the optimized culture conditions for each tumor cell line. This study provides insights into a cell culture technique, which can be optimized for exosome production by various human or mouse tumor cell lines for isolation by mini-SEC.