摘要
外泌体是细胞之间及细胞和细胞外基质之间信号交流的途径之一。近年来,对外泌体的研究呈现显著增长的态势。在体外细胞培养系统中,为保证细胞正常的生理状态,血清是培养液中必须添加的成分。随之而来的问题是,由于血清中含有大量外泌体,其数量数倍于研究中需要进行针对性研究的特定外泌体,从而对特定外泌体的定量和分析造成了干扰。为了去除培养液血清中的外泌体,很多研究者采用差速离心法、密度梯度离心法等进行处理。该研究通过流式细胞分析对外泌体定量后发现,加入2%血清的培养液在进行差速离心处理后,外泌体数量减少了30.6%;而加入5%的血清培养液,外泌体数量下降了32.4%。由此可见,差速离心处理对不同浓度血清的培养液结果类似,只能去除约30%的血清外泌体,留下约70%的血清外泌体,仍然会对实验数据造成干扰。因此,在人脐静脉内皮细胞体外培养系统中,该研究探索了一种新型血清替代培养法,即以无生物成分的血清替代物XerumFree替代培养液中的大部分血清,经过对一系列配比进行探索发现,当XerumFree与胎牛血清FBS体积比为15:1时,细胞既可以正常生长,又可以分泌外泌体,而背景中外泌体干扰程度降低到2%,从而较好地解决了外源性外泌体形成的背景干扰问题,为体外研究外泌体提供了明确的参考方案。
Exosomes are a unique pathway of cell-cell and cell-matrix communication media,which has aroused increasing research interest.In cell culture systems,to maintain normal physiological function of the cell,fetal bovine serum(FBS)is a nearly indispensable additive in the cell culture medium.The consequent problem is that the serum contains a large number of exosomes,several times more than the specific exosomes in the study that need to be targeted,which causes interference in the quantitation and analysis of specific exosomes.Therefore,many studies applied differential centrifugation or density gradient centrifugation to remove the serum exosomes from the culture medium.In this study,we analyzed the decrease percentage of the exosomes from medium with the addition of different amount of FBS by fluorescence activated flow cytometry(FACS).We found that when cell culture medium was supplemented with 2%FBS,the amount of exosome dropped 30.6%after differential centrifugation.When supplemented with 5%FBS,the amount of exosome dropped 32.4%.Differential centrifugation brought no significant difference between the decreasing of the amount of the exosomes in media with different percentages of the FBS,suggesting that the maximum amount of the exosomes can be removed by differential centrifugation is around 30%,while there is still 70%interference remains.Therefore,human umbilical vein endothelial cells(HUVECs)culture system was used to test the minimum FBS to maintain normal cell function and exosome secretion.A serum substitute XerumFree without any biological components was used to replace partial of the FBS.Our results showed that the ratio of 15(XerumFree)to 1(FBS)is the best condition to maintain normal cell function and exosome secretion while the interference of FBS was the minimum(less than 2%).The results from the study provide useful reference and a solid basis to exosome related research.
作者
王丹
韩晓梅
张超
周越
WANG Dan;HAN Xiaomei;ZHANG Chao;ZHOU Yue(School of Biomedical Engineering,Shanghai Jiao Tong University,Shanghai 200030,China)
出处
《生物医学工程学进展》
CAS
2023年第1期42-49,共8页
Progress in Biomedical Engineering
基金
上海市科委“科技创新行动计划”实验动物研究领域项目(16140901900)
关键词
外泌体
细胞培养
差速离心
血清
Exosome
Cell Culture
Differential Centrifugation
Serum
