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Purity and yield of melanoma exosomes are dependent on isolation method.
Journal of Extracellular Vesicles ( IF 16.0 ) Pub Date : 2019-11-20 , DOI: 10.1080/20013078.2019.1692401
Shin La Shu 1 , Yunchen Yang 2 , Cheryl L Allen 1 , Edward Hurley 3 , Kaity H Tung 4 , Hans Minderman 5 , Yun Wu 2 , Marc S Ernstoff 1
Affiliation  

Both exosomes and soluble factors have been implicated in the generation of an immunosuppressive tumour microenvironment. Determining the contribution of each requires stringent control of purity of the isolated analytes. The present study compares several conventional exosome isolation methods for the presence of co-enriched soluble factors while isolating exosomes from human melanoma-derived cell lines. The resultant preparations were analysed by multiplex bead array analysis for cytokine profiles, and by electron microscopy and nanotracking analysis for exosome size distribution and concentration. It is demonstrated that the amount and repertoire of soluble factors in exosome preparations is dependent upon the isolation method used. A combination of ultrafiltration and size exclusion chromatography yielded up to 58-fold more exosomes than ultracentrifugation, up to 836-fold lower concentrations of co-purified soluble factors when adjusted for exosome yield, and a greater than two-fold increase in PD-L1 expressing exosomes. Mechanistically, in context of the immunomodulatory effects of exosomes, the exosome isolation method should be carefully considered in order to limit any effects due instead to co-eluted soluble factors.

中文翻译:

黑色素瘤外泌体的纯度和产量取决于分离方法。

外泌体和可溶性因子都与免疫抑制性肿瘤微环境的产生有关。确定每个的贡献需要严格控制分离分析物的纯度。本研究比较了几种传统的外泌体分离方法,在从人类黑色素瘤衍生细胞系中分离外泌体时,是否存在共富集的可溶性因子。通过多重微珠阵列分析对细胞因子谱分析所得制剂,并通过电子显微镜和纳米追踪分析外泌体大小分布和浓度。结果表明,外泌体制剂中可溶性因子的数量和成分取决于所使用的分离方法。超滤和尺寸排阻色谱的组合产生的外泌体比超速离心多 58 倍,在调整外泌体产量时,共纯化可溶性因子的浓度降低了 836 倍,PD-L1 增加了两倍以上表达外泌体。从机制上讲,在外泌体的免疫调节作用的背景下,应仔细考虑外泌体分离方法,以限制因共洗脱可溶性因子引起的任何影响。
更新日期:2020-04-20
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