关键词: 纳米沟槽, 定向行为, 交流阻抗, 皮肤细胞, 复合型细胞传感器

Cell alignment plays a crucial role in the repair and regeneration of tissues, which is caused by the "contact guidance" of micro/nano structures. In this paper, the nano-grooves with 200 nm in width, 400 nm in period and 75 nm in depth were fabricated on gold substrate with the technique of nanoimprint to simulate the extracellular matrix (ECM). Electric cell-substrate impedance sensing (ECIS) was employed firstly to real-time monitor cell alignment of human foreskin fibroblasts (HFF) and human immortal keratinocyte cells (HaCaT) on nano-grooves, which are two important functional cell types in skin wound-healing. The cell images displayed that the nano-grooves could induce the alignment of HFF cells, in which, the cell arrangement along the direction of nano-grooves occurred prior to the cell elongation. While the nano-grooves couldn't influence the morphology of HaCaT cells, and their adhesion and spread were delayed. In the ECIS monitoring, HFF and HaCaT cells both presented increased normalized impedance (NI) values at their respective characteristic frequencies of 977 and 1465 Hz on nano-grooves and flat electrodes with the prolongation of culture time during 24 h and the increasing trends of NI values were also similar:in the first 6 h, NI values increased faster, and then, the increasing rates declined obviously. HFF cells on nano-grooves generated more intense impedance signals with a larger distinction of increasing rate than those on flat electrodes, indicating that the nano-grooves could promote the adhesion and spread of HFF cells and the directional arrangement had a larger impact on the variation of NI values than cell elongation. While HFF cells adhered and spread in random directions, leading to the reduced difference in increasing rate of NI values. The NI values of HFF cells was further correlated with cell alignment, showing the enhanced impedance responses with the rising percentage of aligned cells. More importantly, there was a good linear correlation between NI values and the percentage of cells arranging along the direction of nano-grooves. In contrast, HaCaT cells had smaller NI values on the nano-grooves with the similar increasing rates, compared to the flat electrodes, revealing that the nano-grooves were less suitable for the adhesion and spread of HaCaT cells with almost no change of cell morphology, and the cell adhesion could cause more obvious variation of NI values than cell spread. Our research would provide a support for the development of complex cell sensors based on ECIS and its application in clinical research field.

Key words: nano-grooves, cell alignment, electric cell-substrate impedance sensing (ECIS), skin cells, complex cell sensor