利用细胞外基质大规模扩增临床级人脂肪间充质干细胞

发布时间：2018-05-28 04:16

本文选题：干细胞 + 脂肪干细胞　；参考：《中国组织工程研究》2014年10期

【摘要】：背景:如何运用无血清培养体系大规模扩增处于未分化状态的脂肪间充质干细胞,并使其保持"干性"是脂肪间充质干细胞临床应用转化中急待解决的难题。目的:建立含细胞外基质的脂肪间充质干细胞体外培养系统,验证其扩增细胞的高效性、有效性及安全性。方法:在无血清培养条件下,将体外分离得到的脂肪间充质干细胞分别接种在包被细胞外基质的培养板和传统二维塑料培养板上。经过体外扩增后,比较2种条件下细胞数量、细胞表面标记物表达、细胞衰老状况以及体外多向分化能力(诱导成脂、成骨和成软骨)的差异,考察脂肪间充质干细胞在含细胞外基质的培养条件下扩增后的临床安全性。结果与结论:脂肪间充质干细胞扩增到第5代时,包被细胞外基质培养板的细胞产量已经是传统二维塑料培养板的10倍以上,流式细胞检测表明,在含细胞外基质条件下扩增的脂肪间充质干细胞仍保持了干细胞表面特定标记物的表达。细胞衰老检测结果显示,使用包被细胞外基质的培养板扩增脂肪间充质干细胞至第15代时,细胞仍几乎无老化现象,而使用二维塑料培养板扩增的细胞在第5代时已出现明显老化,传代后细胞增殖能力明显下降。体外多向诱导分化实验显示,脂肪间充质干细胞在含细胞外基质条件下扩增至第15代时仍具有成脂、成骨和成软骨的分化功能,并且与第5代时没有明显差异,同时显著优于传统培养条件下的第5代脂肪间充质干细胞。染色体核型分析及小鼠成瘤性试验结果表明,脂肪间充质干细胞在含细胞外基质条件下扩增后仍具备临床应用的安全性。以上结果证实,含细胞外基质的无血清培养系统可以更高效且安全地扩增出具有临床应用潜能的脂肪间充质干细胞。
[Abstract]:Background: it is an urgent problem for the clinical application and transformation of adipose mesenchymal stem cells to expand the undifferentiated adipose mesenchymal stem cells in a large scale and keep them dry by using serum-free culture system. Aim: to establish an in vitro culture system of adipose mesenchymal stem cells containing extracellular matrix to verify the efficiency, efficacy and safety of expanded cells. Methods: the adipose mesenchymal stem cells isolated in vitro were inoculated on the culture plate coated with extracellular matrix and on the traditional two-dimensional plastic culture plate under the condition of serum-free culture. After in vitro amplification, the difference of cell number, expression of cell surface markers, cell senescence and the ability of differentiation in vitro (adipogenesis, osteogenesis and cartilage formation) were compared. To investigate the clinical safety of adipose mesenchymal stem cells expanded in extracellular matrix culture. Results and conclusion: when adipose mesenchymal stem cells were expanded to the fifth passage, the cell production of the coated extracellular matrix culture plate was 10 times higher than that of the traditional two-dimensional plastic culture plate. Adipose mesenchymal stem cells containing extracellular matrix maintained the expression of specific markers on the surface of stem cells. The results of cell senescence test showed that the adipose mesenchymal stem cells were expanded with the culture plate coated with extracellular matrix until the 15th generation, the cells still had almost no aging phenomenon. However, the cells amplified by two-dimensional plastic culture plate had obviously aged at the fifth passage, and the proliferation ability of the cells decreased obviously after passage. The multidirectional differentiation experiment in vitro showed that adipose mesenchymal stem cells still had the functions of adipogenesis, osteogenesis and cartilage differentiation at the 15th generation after expansion under the condition of extracellular matrix, and there was no significant difference between adipose mesenchymal stem cells and the fifth generation of adipose mesenchymal stem cells. At the same time, it is superior to the fifth generation adipose mesenchymal stem cells in traditional culture. The results of chromosome karyotype analysis and mouse tumorigenicity test showed that adipose mesenchymal stem cells were still safe for clinical application after expansion under extracellular matrix. These results suggest that the serum-free culture system containing extracellular matrix can effectively and safely amplify adipose mesenchymal stem cells with clinical application potential.
【作者单位】：解放军第455医院解放军南京军区生物细胞工程技术医学转化基地干细胞实验室;上海交通大学医学院附属仁济医院消化科上海市消化疾病研究所干细胞实验室;
【基金】：上海市科委浦江人才计划项目(09PJ1407300) 国家自然科学基金项目(30971468)~~
【分类号】：R329

【共引文献】