Comparison of exosome extracting methods from human umbilical cord mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.0468

Abstract

Abstract:

BACKGROUND: Cell-free stem cell therapy has been an issue of concern, but there is no conclusion on how to extract high-quality exosomes.
OBJECTIVE: To extract exosomes from human umbilical cord mesenchymal stem cells by using three different methods, and then to screen the optimal method.
METHODS: Exosomes were extracted from human umbilical cord mesenchymal stem cells by using the Total Exosome Isolation test kit, Exo Quick test kit and differential ultracentrifugation method, respectively. Then, transmission electron microscopy was used for morphological observations, BCA was utilized to quantify the protein, and western blot assay was applied to detect surface markers CD9, CD81 and CD63.
RESULTS AND CONCLUSION: Extraction of exosomes was completed by all the three methods, and round or oval membranous vesicles were observed under the transmission electron microscope. The protein content and purity of exosomes was highest in the differential ultracentrifugation group, followed by the Exobiology Quick kit group, and lowest in the Total Exosome Isolation kit group, and there were significant differences among the three groups (P < 0.05). Under the same protein concentration, surface specific markers, CD81, CD63 and CD9, were expressed highest in the differential ultracentrifugation group, followed by the Exobiology Quick kit group, and lowest in the Total Exosome Isolation kit group. The operating time was significantly lower in the Exobiology Quick kit group compared with the other two groups (P < 0.05). To conclude, despite a longer operating time, the differential ultracentrifugation method is a rational method to extract enough exosomes with relative high purity.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Exosomes, Umbilical Cord, Mesenchymal Stem Cells, Tissue Engineering

CLC Number:

R394.2

Guo Ying, Wang Xiu-wei, Niu Yu-hu, Wang Li, Zhou Nan, Li Bai-yi, Wang Zhen-dong, Zhang Pin, Gao Ya-jie, Niu Bo. Comparison of exosome extracting methods from human umbilical cord mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(9): 1382-1388.

Figures/Tables 1

2.1 脐带间充质干细胞的形态培养7-12 d时，倒置显微镜下可以看到贴壁细胞，呈梭形，较肥大。待细胞融合率达80%，进行第1次传代，传代培养至第4代，细胞形态均匀一致，排列紧密，集落中心呈漩涡状，见图1。 2.2 细胞表面标志的表达流式细胞仪检测结果显示，细胞高表达CD29、CD44和CD105(> 95%)，极低表达CD34与CD45，符合间充质干细胞的特性，见图2。 2.3 人脐带间充质干细胞的成骨成脂分化能力第4代人脐带间充质干细胞加入成骨诱导培养基后，细胞生长缓慢，培养到第20天经茜素红染色，显微镜下可见有明显的红色钙结节(图3A)。加入成脂诱导培养基后，培养到第15天经油红O染色，镜下可见胞浆内有红色圆形脂滴(图3B)。 2.4 外泌体的形态透射电镜观察发现经3种方法提取到的外泌体均呈椭圆形囊泡，可见外周分界清楚的膜性结构，可单个分布，也可聚集成群。差速超速离心法获得的外泌体直径多分布于30-100 nm，较为均一，Exo Quick试剂盒法获得的外泌体不够均匀，Isolation试剂盒法获得的外泌体背景污染较严重，可能存在蛋白质污染，见图4。 2.5 外泌体蛋白浓度绘制标准曲线，根据标准曲线得出的公式y=0.388x+0.029 4(相关系数R2=0.994 7)分别算出相应的蛋白量，经计算得出差速超速离心法所得的外泌体蛋白浓度为2.0-3.0 g/L，Exo Quick试剂盒提取方法获得的外泌体蛋白浓度为1.5-2.5 g/L，Total Exosome Isolation试剂盒法获得的外泌体蛋白浓度为1.0-2.0 g/L。差速超速离心法获得的外泌体蛋白浓度较其他两种方法高，差异有显著性意义(P < 0.05)。 2.6 SDS-PAGE电泳及凝胶图像 3种方法所得的外泌体中含有多种蛋白成分，而在上样量以及条件相同的情况下，不同方法得到的外泌体的蛋白表达强度不同，在Mr 35 000-49 000之间及Mr19 000以下较为富集。差速超速离心法和Exo Quick试剂盒提取的外泌体在Mr40 000-49 000的蛋白含量明显高于Total Exosome Isolation试剂盒法。在Mr19 000以下，差速超速离心法提取的外泌体蛋白含量明显高于其他两种方法，见图5。 2.7 Western blot检测外泌体标志蛋白表达利用Western blot方法检测3种方法提取的外泌体中的CD9、CD63、CD81表达。结果显示3种方法提取的外泌体都可以表达特异性表面标志物CD9、CD63和CD81(图6)。差速超速离心法提取到的外泌体特异性标志物CD9与CD63在表达强度上远高于其他两种方法；其次，Exo Quick试剂盒法和Total Exosome Isolation试剂盒法提取到的外泌体特异性标志物仅在CD81的表达上略高于差速超速离心法(图7)，说明差速超速离心法提取到的样本较其他两种纯。 2.8 操作时间比较差速超速离心法平均耗时(160.0± 8.0) min；Exo Quick试剂盒法平均耗时(40.0±4.5) min；Total Exosome Isolation试剂盒法平均耗时(111.5±5.0) min。传统差速超速离心法和Total Exosome Isolation试剂盒法的操作时间明显多于Exo Quick试剂盒法，差异有显著性意义 (P < 0.05)。"

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