Effect of 3D-scaffold formation on differentiation and survival in human neural progenitor cells.
Effect of 3D-scaffold formation on differentiation and survival in human neural progenitor cells.
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DOI：
10.1186/1475-925x-9-70
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发表时间：
2010-11-11
影响因子：
3.9
通讯作者：
Frech MJ
中科院分区：
文献类型：
--
作者：
Ortinau S;Schmich J;Block S;Liedmann A;Jonas L;Weiss DG;Helm CA;Rolfs A;Frech MJ
3D支架已被证明可以指导许多不同细胞类型的细胞生长和分化，3D微环境的形成和功能化在决定嵌入细胞的命运方面非常重要。在这里，我们使用了水凝胶为基础的支架，以调查基质浓度和功能化层粘连蛋白对支架的形成的影响，以及这些支架对在其中培养的人神经祖细胞的影响。在这项研究中，我们使用了不同浓度的水凝胶基质PuraMatrix。在一些实验中，我们用层粘连蛋白I功能化基质。用原子力显微镜检查层粘连蛋白的浓度和处理对支架形成的影响。将来自人胎儿神经祖细胞系的细胞在不同基质中以及在2D培养系统中培养，随后用针对神经元标记物的抗体染色进行分析。平行地，通过活/死测定来确定细胞的存活率。原子力显微镜测量表明，矩阵形成的孤立PuraMatrix纤维和纤维聚集体的网络。水凝胶浓度的增加导致支架的网孔尺寸减小，并且用层粘连蛋白进行功能化促进纤维的聚集（束形成），这进一步降低了分离的纤维的密度。我们发现层粘连蛋白功能化对于人类神经祖细胞建立3D生长模式至关重要，并且细胞的增殖也受到基质浓度的影响。此外，我们发现，与2D培养物相比，3D培养物增强了神经元分化和细胞存活率。总之，我们已经证明了3D支架形成对人神经祖细胞的存活和神经元分化的直接影响。这些发现强调了在再生医学背景下，在干细胞和祖细胞体内植入之前优化3D支架方案的重要性。
3D-scaffolds have been shown to direct cell growth and differentiation in many different cell types, with the formation and functionalisation of the 3D-microenvironment being important in determining the fate of the embedded cells. Here we used a hydrogel-based scaffold to investigate the influences of matrix concentration and functionalisation with laminin on the formation of the scaffolds, and the effect of these scaffolds on human neural progenitor cells cultured within them. In this study we used different concentrations of the hydrogel-based matrix PuraMatrix. In some experiments we functionalised the matrix with laminin I. The impact of concentration and treatment with laminin on the formation of the scaffold was examined with atomic force microscopy. Cells from a human fetal neural progenitor cell line were cultured in the different matrices, as well as in a 2D culture system, and were subsequently analysed with antibody stainings against neuronal markers. In parallel, the survival rate of the cells was determined by a live/dead assay. Atomic force microscopy measurements demonstrated that the matrices are formed by networks of isolated PuraMatrix fibres and aggregates of fibres. An increase of the hydrogel concentration led to a decrease in the mesh size of the scaffolds and functionalisation with laminin promoted aggregation of the fibres (bundle formation), which further reduces the density of isolated fibres. We showed that laminin-functionalisation is essential for human neural progenitor cells to build up 3D-growth patterns, and that proliferation of the cells is also affected by the concentration of matrix. In addition we found that 3D-cultures enhanced neuronal differentiation and the survival rate of the cells compared to 2D-cultures. Taken together, we have demonstrated a direct influence of the 3D-scaffold formation on the survival and neuronal differentiation of human neural progenitor cells. These findings emphasize the importance of optimizing 3D-scaffolds protocols prior to in vivo engraftment of stem and progenitor cells in the context of regenerative medicine.