Imaging of Cell-Array by Scanning Electrochemical Microscopy

在本项目中，我们研究了两个主题：1）活细胞的微图案，2）单细胞活性测量。这些研究课题可以组合为“利用微图案化活细胞的生物测定”，这将导致细胞芯片技术的发展。1）细胞微图案的制备：利用PDMS制作的微桩，通过微接触印刷方法成功地在载玻片上制备了粘附性动物细胞的微图案。使用纤连蛋白等细胞粘附蛋白作为墨水材料。HeLa细胞、鸡心肌细胞和PC 12神经元细胞在单细胞水平上成功地形成了细胞网络。应用PEG分子作为细胞电阻修饰效果良好，以保持细胞图案超过5天。2）微图案化细胞的活性评价：通过使用扫描电化学显微镜（SECM）系统对微图案化HeLa细胞的呼吸活性进行成像。结果发现，形状受限细胞的呼吸活性高于自由伸展细胞。3）图案化细胞网络活性的评价：对微图案化心肌细胞进行细胞内Ca ~（2+）显像。微图案化心肌细胞通过形成差距连接形成电共轭网络。在芯片上建立了心肌组织模型，观察了正辛醇、河豚毒素和咖啡因的药理作用。还尝试通过应用微荧光装置系统对图案化细胞进行局部给药。

In the present project, we investigated two topics: 1) the micropatterning of living cells, 2) activity measurements of single cells. These research topics can be combined as the "bioassay using micropatterned living cells", which would lead to the development of cell-chip technology. The results obtained in this project can be summarized as follows,1) Preparation of cellular micropatterns: We succeeded to prepare micropatterns of adhesive animal cells on a glass slides by the micro-contact printing method using PDMS-made microstumps. The cellular adhesive protein such as fibronectin was used as the ink material. HeLa cells, chick cardiomyocytes and PC12 nuronal cells were successfully patterned to form cellular networks on a single cell level. The application of PEG molecules as the cell resistive modification works well so as to maintain the cell pattern for more than 5 days.2) Activity evaluation of the micropatterned cells: The respiratory activity of the micropatterned HeLa cells was imaged by using the scanning electrochemical microscopy (SECM) system. It was found that the respiration activity of the shape-restricted cells is higher than that of the freely spreading cells.3) Evaluation of the activity of the patterned cellular networks: The intracellular Ca2+ imaging was conducted for the micropatterned cardiac myocytes. The micropatterned cardiac myocytes were found to form the electrically conjugated network via forming the gap junctions. It was achieved to observed the pharmacological effects of octanol, tetrodotoxyn, and caffein for the cardiac tissue model on the chip. The local dosing to the patterned cells has also been attempted by applying the microfuluidic device systems.