Zhu Yanxia, Liu Tianqing, Song Kedong, Fan Xiubo, Ma Xuehu, Cui Zhanfeng
Dalian R&D Center for Stem Cell and Tissue Engineering, Dalian University of Technology, China.
Biotechnol J. 2009 Aug;4(8):1198-209. doi: 10.1002/biot.200800130.
Recent reports indicate that adipose tissue is a novel source of multipotent stem cells that can be used in cell therapy and tissue engineering. However, using the traditional cultivation of adipose tissue-derived stem cells (ADSCs), it is hard to meet the needs of clinical applications. To obtain a large number of ADSCs while retaining their stemness, we seeded ADSCs in collagen/chitosan scaffolds and compared the proliferation of ADSCs in a 3-D static environment in dishes and a 3-D dynamic environment in spinner flask. The growth dynamic parameters of ADSCs were examined using a CCK-8 kit every other day, and the variations of glucose and lactic acid concentrations were analyzed every day. After 14 days, the cells were observed under a scanning electron microscope. The surface markers (CD29, CD34, CD44, CD45, CD73, CD105, CD166 and HLA-DR), the specific transcription factors (Nanog, Oct-4, Sox-2 and Rex-1) and the multi-differentiation potential (adipogenic, osteogenic and chondrogenic) were also assayed to identify the stemness of expanded cells. The results showed that the cells in scaffolds in spinner flask could be expanded by more than 26 times, and they presented better morphology and vitality and stronger differentiation ability than the cells cultivated in scaffolds statically. All the cells maintained stem cell characteristics after proliferation. Therefore, spinner flask cultivation is an easy-to-use, inexpensive system for expanding ADSCs in 3-D scaffolds.
最近的报告表明，脂肪组织是多能干细胞的一个新来源，可用于细胞治疗和组织工程。然而，使用传统方法培养脂肪组织来源的干细胞（ADSCs），很难满足临床应用的需求。为了在保留其干性的同时获得大量ADSCs，我们将ADSCs接种在胶原蛋白/壳聚糖支架上，并比较了ADSCs在培养皿中的三维静态环境和转瓶中的三维动态环境中的增殖情况。每隔一天使用CCK-8试剂盒检测ADSCs的生长动力学参数，每天分析葡萄糖和乳酸浓度的变化。14天后，在扫描电子显微镜下观察细胞。还检测了表面标志物（CD29、CD34、CD44、CD45、CD73、CD105、CD166和HLA-DR）、特异性转录因子（Nanog、Oct-4、Sox-2和Rex-1）以及多分化潜能（成脂、成骨和软骨生成），以鉴定扩增细胞的干性。结果表明，转瓶中支架内的细胞可扩增26倍以上，与静态培养在支架内的细胞相比，它们具有更好的形态和活力以及更强的分化能力。所有细胞在增殖后均保持干细胞特性。因此，转瓶培养是一种在三维支架中扩增ADSCs的简便、廉价的系统。
