本文以构建可用于药物开发和评价的细胞芯片实验平台为目标，从细胞培养、细胞给药以及药效评价三个方面开展了一系列研究，包括：（1） 设计、构建并测试了一系列不同类型的基于微电极阵列的介电力辅助的细胞电穿孔芯片，研究了不同类型的芯片结构对细胞介电力捕获和穿孔的影响，并最终优化设计了一款三明治结构的具有选择性细胞定位和电穿孔功能的芯片，实现了单点选通的质粒转染。该方法具有试剂消耗量小、无细胞毒性、使用便捷等优势，为实现高通量全基因组功能分析提供了可靠的实验平台。（2） 利用细胞电阻抗传感技术研究了抗肿瘤药物对人肾小管上皮细胞的毒性作用，用于体外评估抗肿瘤药物的肾毒性，为药物开发中药物肾毒性评价提供了一个有效的筛选工具。此外，我们还尝试将该系统用于评价抗肿瘤药物的解毒剂：硫代硫酸钠和谷胱甘肽对顺铂毒性的缓解作用，所获结果与临床报道一致，提示细胞电阻抗传感技术在解毒剂开发方面的应用价值。（3） 构建了一款将药物浓度梯度生成和细胞电阻抗传感相结合的微流控芯片，实现了不同肿瘤细胞对抗肿瘤药物浓度依赖性和药物敏感性的实时检测。该芯片实现了一款新颖的被动式“气泡阀”来实现浓度梯度的维持。与传统的气动阀相比，该“气泡阀”具有结构简单，使用方便，无需外界控制系统的优势。（4） 提出了一种将传统铸模和气体铸模相结合的快速构建含三维结构的微器件的方法。该方法能在单次注塑的过程中同时构建凹凸两种类型的结构，分别用于细胞捕获和管道形成。通过该方法我们构建了具有凹坑阵列的流体芯片，可用于形成尺寸均一可控的拟胚体，为后续的干细胞分化研究打下了基础。该三维培养方法有潜力为药物开发提供更接近于生理状况的实验平台。
The goal of this dissertation is to develop cell chips for drug discovery and evaluation. This dissertation mainly focuses on the chip-based techniques about cell culture, transfection and monitoring with the following achievement accomplished.Firstly, we have constructed and optimized a series of microelectrode array based chips for dielectrophoresis assisted electroporation (EP), and examined the influence of chip designs upon the efficiency of cell trapping and EP. We selected an optimized chip with sandwiched structure possessing functions of selective cell patterning and EP and finally achieved the individually addressable plasmids transfer. The approach presented here has advantages such as low reagent consumption, non-cytotoxicity and flexibility, which provides a valuable tool for functional studies of genes.Secondly, we have studied the anti-cancer drug induced apoptosis in human kidney cell to predict drug nephrotoxicity using cellular impedance sensing. The impedance data obtained in these experiments agreed well with the results obtained using MTT and flow cytometry, and correlated well with clinical research results. Furthermore, we investigated the inhibition effect of antidotes of DDP: glutathione (GLU) and sodium subsulfite (STS), which decreased the nephrotoxicity of DDP in a dose dependent manner. It presents a real-time system for nephrotoxicity and antidotes of anti-cancer drugs assessment in vitro, which is easy to use and may become a tool of great potential for drug evaluation.Thirdly, we have integrated the cellular impedance sensing with the chemical concentration generator to construct a microfluidic device facilitating the monitoring of the cytotoxicity of an anti-cancer drug in different cancer cell lines. A novel passive "air-bubble" valve was introduced into this device allowing the automatic closure of the valve without the need of any external valve-control instrument to maintain the static concentration gradients. Compared to the traditional pneumatic microvavles, our air-bubble valve is simpler and more flexible.Finally, we have proposed a novel method for the fabrication of polydimethylsiloxane (PDMS) microdevices with complicated 3-D structures, such as concave and crater shapes, using an easily machined polymethyl methacrylate (PMMA) mold combined with a one-step molding process consisting of replica molding and air molding. The procedure presented here enables rapid preparation of protruding and concave microstructures varying in shapes and dimensions on the same substrate, which allows for more flexible device design and applications. We have also fabricated a fluidic device with an array of concave shaped-microwells to regulate the size of embryoid bodies, and satisfactory results were obtained. This method provides a 3-D cell culture system for drug screening.
