海藻酸钙微胶珠培养神经干细胞研究

Culture of Neural Stem Cells in Alginate Microbeads

神经干细胞作为一种具备自我更新及多向分化潜能的细胞,已经引起国内外研究者的高度关注。神经干细胞的发现、研究与应用,将在治疗神经系统疾病以及神经损伤的修复中起到重要作用,但是神经干细胞的来源受限及数量不足是临床应用的最大障碍。采用三维动态培养从而实现神经干细胞大规模扩增是解决其数量不足的有效方法。但是,在三维动态搅拌条件下,剪切力对细胞的损害是非常大的。考虑到海藻酸钙微胶珠在细胞培养领域的应用,如果把神经干细胞包囊到微胶珠中进行动态培养,将会避免剪切力对干细胞的损伤,因此是一个非常值得期待的干细胞培养方式。本研究在静态条件下,采用海藻酸钙微胶珠作为神经干细胞的3D培养手段,对成囊工艺进行了探索,以确定适于培养神经干细胞的最佳胶珠制备工艺。通过微胶珠内部的扩散传质模型,研究了微胶珠制备工艺中各参数对其扩散系数(D)的影响,找出微胶珠最佳制备工艺参数为:直径2mm,1.5%(wt%)海藻酸钠滴入3.5%(wt%)CaCl2中,反应时间10min。确定上述工艺后,对小鼠神经干细胞进行了不同包囊密度条件下的培养,得到包囊神经干细胞的最佳密度为0.08×106cells.mL-1。此外,本研究还对海藻酸钙微胶珠培养的神经干细胞进行了nestin等特异性蛋白的免疫荧光化学染色,结果为阳性,表明所培养的神经干细胞能够保持其干细胞的特性。本研究结果表明,采用优化参数制备的海藻酸钙微胶珠是培养神经干细胞的有效手段。同时也为动态微囊培养神经干细胞打下了良好基础。

Neural Stem Cells （NSCs） with the capacity of self-renewal and differentiation into neurons and glial ceils have obtained more and more worldwide attention. The finding, research and application of NSCs will play an important role in the nerve disease treatment and nerve injury repair. The shortage of the source and the amount of NSCs, however, is the main obstacle for its clinic application. Large-scale three-dimensional culture of NSCs in vitro provides a feasible answer for this problem. But the shear stress in bioreactors would cause serious cell damage especially for the shear sensitive cells like NSCs. To avoid the shear stress, encapsulation of NSCs and then cultivation in bioreactors is thus worth of investigating. Therefore, we explored the method of culturing NSCs in Calcium-Alginate Microbeads （Ca-Alg MBs）. To provide the optimum growth conditions for NSCs in Ca-Alg MBs, the gelation parameters, such as diameter, reactant concentration, and gelation time were determined firstly to form the proper bead structure for cell growth. The diffusion experiment was performed to get the diffusion data for different Ca-Alg MBs and a diffusive mathematical model was set up to find the diffusion coefficient （D） of glucose in Ca-Alg MBs by fitting the experimental data. Base on the orthogonal test, the optimum gelation conditions for preparing microbeads with a proper diffusivity were determined. And the gelation parameters were 2mm in bead diameter, 1.5% sodium alginate, 3.5%CaCl2 , and the gelation time of 10 min. The culture of encapsulated NSCs with different cell density was then conducted. The results showed that the culture effect with the encapsulation density of 0.08 × 10^6cells· mL^-1 was the best among the testing samples. The recovered NSCs were stained by immunofluoreseenee and the majority of the expanded NSCs in Ca-Alg MBs were nestin-positive. The experimental results demonstrated that mouse hippocampus-defived NSCs could survive and be expanded in Calcium-alginate Mierobeads.