Department of Materials Science and Engineering, Indian Institute of Technology, Kanpur 208016, India; Materials Research Centre, Indian Institute of Science, Bangalore 560012, India.
Biomaterials. 2013 Dec;34(37):9252-63. doi: 10.1016/j.biomaterials.2013.08.057. Epub 2013 Sep 10.
We demonstrate the efficacy of amorphous macroporous carbon substrates as electrodes to support neuronal cell proliferation and differentiation in electric field mediated culture conditions. The electric field was applied perpendicular to carbon substrate electrode, while growing mouse neuroblastoma (N2a) cells in vitro. The placement of the second electrode outside of the cell culture medium allows the investigation of cell response to electric field without the concurrent complexities of submerged electrodes such as potentially toxic electrode reactions, electro-kinetic flows and charge transfer (electrical current) in the cell medium. The macroporous carbon electrodes are uniquely characterized by a higher specific charge storage capacity (0.2 mC/cm(2)) and low impedance (3.3 kΩ at 1 kHz). The optimal window of electric field stimulation for better cell viability and neurite outgrowth is established. When a uniform or a gradient electric field was applied perpendicular to the amorphous carbon substrate, it was found that the N2a cell viability and neurite length were higher at low electric field strengths (≤ 2.5 V/cm) compared to that measured without an applied field (0 V/cm). While the cell viability was assessed by two complementary biochemical assays (MTT and LDH), the differentiation was studied by indirect immunostaining. Overall, the results of the present study unambiguously establish the uniform/gradient vertical electric field based culture protocol to either enhance or to restrict neurite outgrowth respectively at lower or higher field strengths, when neuroblastoma cells are cultured on porous glassy carbon electrodes having a desired combination of electrochemical properties.
我们证明了无定形大孔碳基底作为电极在电场介导的培养条件下支持神经元细胞增殖和分化的功效。电场垂直于碳基底电极施加，同时在体外培养小鼠神经母细胞瘤（N2a）细胞。第二个电极放置在细胞培养液之外，允许在不考虑淹没电极的复杂情况（例如潜在的有毒电极反应、电动流和细胞培养液中的电荷转移（电流））的情况下研究细胞对电场的反应。大孔碳电极的独特特点是具有更高的比电荷存储容量（0.2 mC/cm²）和低阻抗（1 kHz 时为 3.3 kΩ）。建立了最佳的电场刺激窗口，以获得更好的细胞活力和突起生长。当垂直于无定形碳基底施加均匀或梯度电场时，与没有施加电场（0 V/cm）时相比，在较低的电场强度（≤2.5 V/cm）下，N2a 细胞活力和突起长度更高。虽然细胞活力通过两种互补的生化测定法（MTT 和 LDH）进行评估，但分化通过间接免疫染色进行研究。总的来说，本研究的结果明确确立了基于均匀/梯度垂直电场的培养方案，当神经母细胞瘤细胞在具有所需电化学性能组合的多孔玻璃状碳电极上培养时，在较低或较高场强下分别增强或限制突起生长。
