脂肪干细胞的三维球形培养

发布时间：2018-10-25 17:56

【摘要】：背景:大多哺乳类细胞在正常生理状态下多以三维结构存在,为还原细胞本身在体内的自然状态,很多研究者开始尝试在体外对细胞进行三维培养,球形培养是一种常见的三维培养模式。目的:尝试用3种不同的方法在体外对人脂肪干细胞进行球形培养,并观察其生物学特征。方法:对提取的脂肪来源细胞进行表面Marker流式检测以及成脂成骨诱导鉴定后,证实为脂肪干细胞。先后用低黏附培养法、hanging-drop培养法和Eppendorf管培养法3种方法在体外对脂肪干细胞进行球形培养,对3种方法形成球形的特点进行比较分析,并通过Imagej软件计算出3组多细胞球体的平均面积,利用Viability/Cytotoxicity Assay Kit for Animal LiveDead Cells试剂盒对3组多细胞球体进行活力检测。结果与结论:①通过流式细胞术鉴定细胞表型标志物,其中CD29,CD44,CD59完全阳性,同时成脂成骨诱导也为阳性,证实提取的细胞为脂肪干细胞。3代以内脂肪干细胞多呈长梭形,并克隆状生长。②低黏附培养法、hanging-drop培养法、Eppendorf管培养法均可使脂肪干细胞聚集成球形生长,但所成多细胞球形有所差异。低黏附培养法所形成的细胞球形大小不一,不易控制;而hanging-drop培养法和Eppendorf管培养法均可使脂肪干细胞形成大小均一的球形,但在大小和形成时间上有所不同。③3种不同方法所形成的球形细胞均保持较好的细胞活力。
[Abstract]:Background: most mammalian cells in normal physiological state exist in a three-dimensional structure. In order to reduce the natural state of the cells in vivo, many researchers began to try to culture the cells in vitro. Spherical culture is a common three-dimensional culture model. Aim: to study the biological characteristics of human adipose stem cells cultured in vitro with three different methods. Methods: the extracted adipose derived cells were confirmed as adipose stem cells by surface Marker flow detection and adipogenic osteogenesis induction. Three methods of low adhesion culture, hanging-drop culture and Eppendorf tube culture were used to culture adipose stem cells in vitro. The mean area of three groups of multicellular spheres was calculated by Imagej software, and the viability of the three groups of multicellular spheres was detected by Viability/Cytotoxicity Assay Kit for Animal LiveDead Cells kit. Results and conclusion: 1 the phenotypic markers were identified by flow cytometry, in which CD29,CD44,CD59 was completely positive and adipogenic osteogenesis was positive, which confirmed that the extracted cells were adipose stem cells. 2 low adhesion culture method, hanging-drop culture method and Eppendorf tube culture method can make adipose stem cells aggregate into globular growth, but there are some differences in the formation of multicellular spheres. The spherical cells formed by low adhesion culture are different in size and difficult to control, but both hanging-drop culture and Eppendorf tube culture can make adipose stem cells to form uniform globules. However, the size and time of formation were different, and the spherical cells formed by 33 different methods kept good cell viability.
【作者单位】：暨南大学再生医学教育部重点实验室;暨南大学医学院眼科研究所;
【基金】：国家自然科学基金项目“微重力介导角膜基质细胞的iPS诱导及其角膜内皮细胞分化(30973244)”~~
【分类号】：R329.2

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