神经干细胞体外三维培养及其模型研究
发布时间：2018-09-19 07:24
【摘要】： 神经干细胞(Neural stem cells,NSCs)作为一种具备自我更新能力及多向分化潜能的细胞,自被发现以来,就因其对神经系统的修复能力而受到广大学者的关注。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。采用体外培养以实现大规模扩增是这个问题的一种有效解决办法。然而,在二维神经球悬浮培养条件下,由于球体自身结构的制约,随着神经球的直径增长,营养物质由外及内传递以及细胞代谢物由内及外扩散均受到影响。考虑到生物支架材料-胶原(Collagen I,COL I)在干细胞体外大规模培养领域的应用,如果把细胞接种在由胶原制成的三维支架材料中进行培养,将会成为一个更有效的神经干细胞培养方式。 为了深入了解神经干细胞在由胶原制成的三维支架材料中的生长状况,本文将实验数据与传质代谢方程相结合,通过求解数学模型,计算支架内各种代谢物质的浓度分布,进而筛选出最有利于培养神经干细胞的胶原支架厚度和适宜的细胞接种密度。 首先,从E14小鼠胚胎海马脑组织中获得原代细胞,体外无血清悬浮培养,利用诱导分化和免疫荧光对传代细胞进行生物学鉴定,同时测定单个神经干细胞对溶解氧的代谢参数。其次,检测细胞-胶原支架的葡萄糖、乳酸代谢情况,与传质代谢模型计算结果进行比对,验证模型的可靠性。最后,通过计算不同厚度胶原支架内代谢物质的浓度分布,选择最佳成胶工艺参数。 实验结果表明:①所培养的细胞具有增殖能力和分化潜能,符合神经干细胞的定义,可用于单个神经干细胞氧代谢的研究。②对所培养的神经干细胞氧代谢的测定结果显示,单个神经干细胞的氧代谢速率为2.9×10~(-14)mmol/cell/s。③胶原的终浓度不宜低于1mg/ml,否则无法满足三维支架制备过程中对机械强度的要求。④在葡萄糖初始浓度5.08×10~(-2)mmol/ml、培养基液层厚度5mm、细胞接种密度1×10~6cells/ml为培养条件下,2mm厚度的胶原支架完全能够满足细胞正常生长对葡萄糖、乳酸和溶解氧浓度的要求。 结合模型计算,最终确定神经干细胞接种于胶原支架内进行体外三维培养的适宜条件为:凝胶层厚度2mm、培养基液层厚度5mm、细胞接种密度2×10~5cells/ml。
[Abstract]:Neural stem cell (Neural stem cells,NSCs), as a kind of cells with self-renewal ability and multi-differentiation potential, has attracted much attention since it was discovered because of its ability to repair the nervous system. However, limited sources and insufficient number of neural stem cells are the biggest obstacle to clinical application. In vitro culture for large-scale amplification is an effective solution to this problem. However, under the condition of 2-D suspension culture, due to the restriction of the structure of the sphere itself, with the increase of the diameter of the ball, the transfer of nutrients from the outside and the diffusion of the metabolites from the inside and outside are affected. Considering the application of biological scaffold collagen (Collagen I) in the field of stem cell mass culture in vitro, if the cells were cultured in a three-dimensional scaffold made of collagen, It will be a more effective way to culture neural stem cells. In order to understand the growth of neural stem cells (NSCs) in three dimensional scaffolds made of collagen, this paper combines the experimental data with the mass transfer equation, and calculates the concentration distribution of various metabolites in the scaffold by solving the mathematical model. The thickness of collagen scaffold and the suitable seeding density were selected. First, primary cells were obtained from the hippocampus of E14 mouse embryos, and cultured without serum in vitro. The passage cells were identified by induction differentiation and immunofluorescence, and the metabolic parameters of dissolved oxygen by single neural stem cells were measured. Secondly, the glucose and lactate metabolism of the cell-collagen scaffold was detected and compared with the results of mass transfer model to verify the reliability of the model. Finally, by calculating the concentration distribution of metabolites in collagen scaffolds with different thickness, the optimum gelling process parameters were selected. The results show that the cells cultured at 1: 1 have the ability of proliferation and differentiation, which accord with the definition of neural stem cells, and can be used in the study of oxygen metabolism of single neural stem cells. The oxygen metabolism rate of single neural stem cells is 2.9 脳 10 ~ (-14) mmol/cell/s.3 collagen concentration should not be less than 1 mg / ml, otherwise, it can not meet the requirement of mechanical strength in the process of three-dimensional scaffold preparation. 4 at the initial glucose concentration of 5.08 脳 10 ~ (-2) mmol/ml, medium, the thickness of the liquid layer is 5 mm and the thickness is fine. The collagen scaffold with a thickness of 2 mm and a cell density of 1 脳 10~6cells/ml could completely satisfy the normal growth of the cells to glucose. Lactic acid and dissolved oxygen concentration requirements. Combined with the model calculation, the suitable conditions for the three-dimensional culture of neural stem cells in collagen scaffold were determined as follows: the thickness of gel layer was 2 mm, the thickness of medium was 5 mm, and the cell density was 2 脳 10 ~ (5) cells / ml.
【学位授予单位】：大连理工大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
本文编号：2249436
[Abstract]:Neural stem cell (Neural stem cells,NSCs), as a kind of cells with self-renewal ability and multi-differentiation potential, has attracted much attention since it was discovered because of its ability to repair the nervous system. However, limited sources and insufficient number of neural stem cells are the biggest obstacle to clinical application. In vitro culture for large-scale amplification is an effective solution to this problem. However, under the condition of 2-D suspension culture, due to the restriction of the structure of the sphere itself, with the increase of the diameter of the ball, the transfer of nutrients from the outside and the diffusion of the metabolites from the inside and outside are affected. Considering the application of biological scaffold collagen (Collagen I) in the field of stem cell mass culture in vitro, if the cells were cultured in a three-dimensional scaffold made of collagen, It will be a more effective way to culture neural stem cells. In order to understand the growth of neural stem cells (NSCs) in three dimensional scaffolds made of collagen, this paper combines the experimental data with the mass transfer equation, and calculates the concentration distribution of various metabolites in the scaffold by solving the mathematical model. The thickness of collagen scaffold and the suitable seeding density were selected. First, primary cells were obtained from the hippocampus of E14 mouse embryos, and cultured without serum in vitro. The passage cells were identified by induction differentiation and immunofluorescence, and the metabolic parameters of dissolved oxygen by single neural stem cells were measured. Secondly, the glucose and lactate metabolism of the cell-collagen scaffold was detected and compared with the results of mass transfer model to verify the reliability of the model. Finally, by calculating the concentration distribution of metabolites in collagen scaffolds with different thickness, the optimum gelling process parameters were selected. The results show that the cells cultured at 1: 1 have the ability of proliferation and differentiation, which accord with the definition of neural stem cells, and can be used in the study of oxygen metabolism of single neural stem cells. The oxygen metabolism rate of single neural stem cells is 2.9 脳 10 ~ (-14) mmol/cell/s.3 collagen concentration should not be less than 1 mg / ml, otherwise, it can not meet the requirement of mechanical strength in the process of three-dimensional scaffold preparation. 4 at the initial glucose concentration of 5.08 脳 10 ~ (-2) mmol/ml, medium, the thickness of the liquid layer is 5 mm and the thickness is fine. The collagen scaffold with a thickness of 2 mm and a cell density of 1 脳 10~6cells/ml could completely satisfy the normal growth of the cells to glucose. Lactic acid and dissolved oxygen concentration requirements. Combined with the model calculation, the suitable conditions for the three-dimensional culture of neural stem cells in collagen scaffold were determined as follows: the thickness of gel layer was 2 mm, the thickness of medium was 5 mm, and the cell density was 2 脳 10 ~ (5) cells / ml.
【学位授予单位】：大连理工大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
【引证文献】
相关硕士学位论文 前1条
1 吕艳玲;胚胎大鼠海马神经干细胞的扩增及其与三维微小凹图式复合的研究[D];重庆大学;2010年
，本文编号：2249436
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