Strong attachment of circadian pacemaker neurons on modified ultrananocrystalline diamond surfaces.
Strong attachment of circadian pacemaker neurons on modified ultrananocrystalline diamond surfaces.
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DOI：
10.1016/j.msec.2016.03.092
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发表时间：
2016-07
期刊：
影响因子：
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通讯作者：
A. Voss;Hongying Wei;Yi Zhang;S. Turner;G. Ceccone;J. Reithmaier;M. Stengl;C. Popov
中科院分区：
文献类型：
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作者：
A. Voss;Hongying Wei;Yi Zhang;S. Turner;G. Ceccone;J. Reithmaier;M. Stengl;C. Popov
金刚石是一种有前景的材料，可用于许多生物应用，包括制造神经元和神经假体（例如视网膜植入物）的附着和研究平台。目前的工作是通过微波等离子体化学气相沉积法沉积超纳米晶金刚石 (UNCD) 薄膜，并通过 UV/O3 处理或 NH3 等离子体进行改性，并全面表征其体积和表面特性，如结晶度、形貌、成分和化学键合性质。研究了昆虫昼夜节律起搏器神经元与带有 H-、O- 和 NH2- 末端的 UNCD 表面的细胞密度和活力的相互作用。无需应用粘附蛋白即可实现快速且牢固的附着，从而可以对用于制备原代细胞培养物的分散方案进行有利的修改。用于将分散的细胞颗粒化以将其与分散酶分离的离心步骤很容易损伤神经元。现在可以避免离心，因为分散的神经元快速而牢固地附着在 UNCD 表面上。酶溶液可以很容易地洗掉，而不会损失许多分散的细胞。钙成像显示，没有观察到对细胞活力和生理反应的不利影响。此外，神经元的附着增强，特别是在修饰的UNCD表面上，对于细胞培养物的免疫细胞化学程序特别有利。与对照相比，洗涤步骤期间的细胞损失显着减少一个数量级。此外，UNCD 薄膜下钛网格结构的集成允许将生理特征神经元单独分配给免疫细胞化学染色的细胞。因此，采用不含外来蛋白质的UNCD表面改善了细胞培养方案和培养细胞的免疫细胞化学。神经元快速而牢固的附着归因于地形、表面化学和润湿性的有利组合。
Diamond is a promising material for a number of bio-applications, including the fabrication of platforms for attachment and investigation of neurons and of neuroprostheses, such as retinal implants. In the current work ultrananocrystalline diamond (UNCD) films were deposited by microwave plasma chemical vapor deposition, modified by UV/O3treatment or NH3plasma, and comprehensively characterized with respect to their bulk and surface properties, such as crystallinity, topography, composition and chemical bonding nature. The interactions of insect circadian pacemaker neurons with UNCD surfaces with H–, O– and NH2-terminations were investigated with respect to cell density and viability. The fast and strong attachment achieved without application of adhesion proteins allowed for advantageous modification of dispersion protocols for the preparation of primary cell cultures. Centrifugation steps, which are employed for pelletizing dispersed cells to separate them from dispersing enzymes, easily damage neurons. Now centrifugation can be avoided since dispersed neurons quickly and strongly attach to the UNCD surfaces. Enzyme solutions can be easily washed off without losing many of the dispersed cells. No adverse effects on the cell viability and physiological responses were observed as revealed by calcium imaging. Furthermore, the enhanced attachment of the neurons, especially on the modified UNCD surfaces, was especially advantageous for the immunocytochemical procedures with the cell cultures. The cell losses during washing steps were significantly reduced by one order of magnitude in comparison to controls. In addition, the integration of a titanium grid structure under the UNCD films allowed for individual assignment of physiologically characterized neurons to immunocytochemically stained cells. Thus, employing UNCD surfaces free of foreign proteins improves cell culture protocols and immunocytochemistry with cultured cells. The fast and strong attachment of neurons was attributed to a favorable combination of topography, surface chemistry and wettability.