Oriented immobilization of epidermal growth factor onto culture substrates for the selective expansion of neural stem cells
Oriented immobilization of epidermal growth factor onto culture substrates for the selective expansion of neural stem cells
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DOI：
10.1016/j.biomaterials.2007.04.022
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发表时间：
2007-08-01
期刊：
影响因子：
14
通讯作者：
Iwata, Hiroo
中科院分区：
文献类型：
--
作者：
Nakaji-Hirabayashi, Tadashi;Kato, Koichi;Iwata, Hiroo
为了开发允许神经干细胞（NSC）有效扩增的培养基质，通过使用重组技术将Ni（II）配位至融合至EGF的C末端的组氨酸标签，将表皮生长因子（EGF）固定至基于玻璃的基质的Ni（II）螯合表面上。为了制备镍螯合表面，将薄的金层沉积到玻璃表面。然后在金表面形成三价羧酸封端的烷基硫醇自组装膜，并与Ni（II）离子形成螯合物。在单层的制备中，将三甘醇封端的烷醇与羧酸封端的烷醇以各种组合物混合，以减少EGF的非特异性吸附。通过X射线光电子能谱、红外反射-吸收光谱和接触角测量对单分子膜进行表面分析。进行表面等离子体共振分析和蛋白质测定以表征EGF固定的表面。在EGF螯合的底物上检测大鼠胎儿NSC的增殖和分化，以定量评估烷醇组合物对干细胞扩增效率的影响。结果表明，扩增效率依赖于烷基硫醇的组成。这一结果可归因于螯合EGF的表面密度的差异。在最佳条件下，98%的增殖细胞表达NSC标记。此外，这些细胞可以传代培养以进一步扩增，同时保留其多能性。我们的结论是，在这里开发的基板提供了高选择性扩增神经干细胞的有效方法。(c)2007爱思唯尔有限公司保留所有权利。
To develop a Culture Substrate that allows efficient expansion of neural stem cells (NSCs), epidermal growth factor (EGF) was immobilized onto the Ni(II)-chelated surface of a glass-based substrate through coordination of Ni(II) to the histidine tag that was fused to the C-terminal of EGF using recombinant technology. For the preparation of the nickel-chelated surface, a thin gold layer was deposited to the glass surface. and then the self-assembled monolayer of alkanethiol terminated with trivalent carboxylic acids was formed on gold and chelated with Ni(II) ions. In the preparation of a monolayer, triethylene glycol-terminated alkanethiol was mixed with carboxylic acid-terminated alkanethiol at various compositions in order to reduce the non-specific adsorption of EGF. The surface analysis of the monolayers was performed by X-ray photoelectron spectroscopy, infrared reflection-absorption spectroscopy, and contact angle measurements. surface plasmon resonance analyses and protein assays were performed for characterizing EGF-immobilized surfaces. The proliferation and differentiation of rat fetal NSCs were examined on the EGF-chelated substrates to assess quantitatively the effects of alkanethiol composition on the efficiency of stem cell amplification. It was shown that the amplification efficiency was dependent on the alkanethiol composition. This result could be attributed to the difference in the surface density of chelated EGF. Under the optimal condition, 98% of proliferated cells expressed NSC marker. In addition, these cells could be subcultured for further expansion, while retained their multipotency. We concluded that the substrate developed here provides the efficient method for the highly selective expansion of NSCs. (c) 2007 Elsevier Ltd. All rights reserved.