培养基中细胞生长因子增强人间充质干细胞成瘤性风险  

Growth factors in a culture medium may enhance tumorigenic potency of human mesenchymal stem cells

作　　者：张可华 贾春翠 吴雪伶 纳涛 韩晓燕 吴婷婷 孟淑芳 ZHANG Ke-hua;JIA Chun-cui;WU Xue-ling;NA Tao;HAN Xiao-yan;WU Ting-ting;MENG Shu-fang(Cell Collection and Research Center,Institutes for Biological Products Control,National Institutes for Food and Drug Control,Beijing 100050,China)

机构地区：[1]中国食品药品检定研究院生物制品检定所细胞资源保藏研究中心,100050

出　　处：《中国医药生物技术》2021年第6期481-491,共11页Chinese Medicinal Biotechnology

基　　金：中国科学院战略性先导科技专项(XDA16040502);中国药品监管科学行动计划细胞和基因治疗产品技术评价与监管体系研究。

摘　　要：目的本中心在人间充质干细胞(hMSCs)第三方复核检验中发现某些无血清培养基会促使hMSCs在软琼脂克隆试验中呈现阳性结果。为进一步明确该现象,探索促使hMSCs软琼脂克隆阳性的培养基成分并深入分析其风险。方法比较几种不同品牌的商品化无血清培养基对hMSCs软琼脂克隆形成的影响,通过细胞因子抗体芯片和ELISA定量检测筛选并初步确定促使软琼脂克隆形成的培养基成分,通过在培养基中添加特定成分进一步明确该成分对hMSCs成瘤性的影响,并通过长期添加试验和细胞转录组测序分析该培养基成分持续作用对hMSCs生长和基因转录方面的影响。结果研究结果显示,部分品牌无血清培养基可促使hMSCs在软琼脂克隆形成试验中呈现阳性。培养基成分分析显示这些培养基中表皮细胞生长因子(EGF)、碱性成纤维细胞生长因子(bFGF)和血小板源性生长因子(PDGF-BB)呈现出较高水平。在含10%FBS的完全培养基中分别或组合添加EGF(50 ng/ml)、bFGF(50 ng/ml)和PDGF-BB(10 ng/ml),均可促使hMSCs软琼脂克隆形成。以上述浓度添加生长因子持续培养hMSCs 10代后,再撤掉生长因子继续培养3代,细胞增殖和基因转录发生显著改变。结论培养基中含有的高浓度EGF、bFGF和PDGD-BB等生长因子成分能够增强hMSCs成瘤性风险,用这些生长因子长期培养hMSCs可对细胞增殖和基因转录造成显著影响。本研究提示在hMSCs类细胞治疗产品工艺研发中,需高度关注培养基关键成分对hMSCs特性的影响,结合成瘤性分析优化并筛选最适培养基。Objective In the third-party review of human mesenchymal stem cells(hMSCs),some serum-free mediums were found to promote colony formation of hMSCs in soft agar gels.In order to further clarify this phenomenon,this study was conducted to explore the components of the medium that led to the positive cloning of hMSCs soft agar and to further analyze their risks.Methods Colony formation abilities were compared between hMSCs grown under different brands of commercial serum-free medium.The components of these culture media that promoted the colony formation of cells were screened and determined by cytokine antibody chip assay and ELISA.Tumor promotion ability of these components was further confirmed by adding these specific components to the medium.Long term effects of these components on the growth and gene transcription of hMSCs were further clarified through culturing cells for a long period with those gradients.Results This study confirmed that some brands of serum-free medium could promote the colony formation of hMSCs in soft agar.The data from medium composition analysis showed the high levels of epidermal growth factor(EGF),basic fibroblast growth factor(bFGF)and platelet-derived growth factor(PDGF-BB)in these media.Adding EGF(50 ng/ml),bFGF(50 ng/ml)and PDGF-BB(10 ng/ml)to the complete medium containing 10%FBS could induce the colony formation of hMSCs in soft agar.The proliferation and gene transcription of hMSCs were significantly changed after 10 passages of culture with the above concentration of growth factors and additional 3 passages of culture without these growth factors.Conclusion High concentration of growth factors,such as EGF,bFGF,and PDGF-BB,in cell culture medium can enhance the tumorigenicity risk of hMSCs.Long term culture of hMSCs with these growth factors can cause significant effects on cell proliferation and gene expression profiles.This study suggests that the researchers and developers of hMSCs products should pay close attention in the critical components in cell culture media and anal

关 键 词：间充质干细胞 成瘤性 表皮细胞生长因子 成纤维细胞生长因子 血小板源性生长因子 软琼脂克隆形成试验 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]