Study Of A Microelectrode Array Based High-throughput Cell-electrofusion Method

Since cell-electrofusion technology was invented by Ulrich Zimmermann, due to its many advantages such as high efficiency, good controllability, simple operation, excellent repeatability and harmlessness to cells, it has been extensively applied in species production or improvement, monoclonal antibody preparation, immunological treatment of cancer, and so on.This thesis is involved in the research and development of a microelectrode array based cell-electrofusion chip on about 1 cm2 substrate by using MEMS manufacturing technology. The number of microelectrode pairs on this chip reaches the magnitude of 103 and the distance between each couple of electrodes is less than 100μm. Based on the study, a cell-electrofusion instrument was developed, which was integrated with the chip to form a high-throughput cell-electrofusion system. Using the principles of dielectrophoresis and electroporation, the instrument can be used to manipulate cells and carry out cell fusion.Cell location, electroporation and extrusion are the bases for cell fusion. Therefore, at first our study focused on exploring the principles of cell manipulation and cell fusion. Based on theoretical study, finite element method was used to analyze the profile of electric field within a microelectrode array with different shape, position, and external voltage applied. The computational result can be used to optimize the design of microelectrode array in order to obtain a favorable electric field environment. Based on the analysis, an interdigital, pectinate, and rectangular microelectrode array model was chosen. Then, studies have been done about the material choice, chip processing technology, and packaging technology, etc. And as a result, four cell-electrofusion chips fabricated on different materials, i.e. silicon-silicon, glass-silicon, silicon-metal and PCB, were developed. Meanwhile, high-throughput cell-electrofusion instrument was developed. Finally, cell-fusion experiments on microbiologic cells, animal cells, and plant cells were carried out. The detailed study includes:1. The impact of the size and distribution of microelectrode on the electric-field intensity and gradient. On the cell-electrofusion chip, the 3-D microelectrode geometry and distribution parameters include the thickness, width, and depth of the electrode, and the longitudinal and lateral interval between electrode pairs, the geometry shape (right angled tooth, cone-shape tooth, pillbox) of the tip of the microelectrode, and the distribution (symmetry and asymmetrical) of a microelectrode array, etc. The change of these parameters will influence the electric-field intensity and the gradient in microchannel. Based on the finite element method, modeling and simulation of the microelectrode array were carried out. According to the requirement of cell electrofusion, the interdigital, pectinate, and rectangular microelectrode array was chosen for high-efficiency cell electrofusion. The chip can produce a suitable electric field environment for cell electrofusion and enhance the controlling ability for cells and the fusion efficiency.2. Choice of the material and fabrication technology for the chip and microelectrode. For obtaining best electrical and biochemical performance, four types of chips with different electrode were studied, including silicon-silicon, glass-silicon, silicon-metal and PCB microelectrodes. This thesis also studied the design of the microelectrode and microchannel, fabrication technique, material choice, and packaging technology, etc.3. Design and manufacture of cell-electrofusion instrument. After studying the electric signal characteristics required in cell alignment, electroporation and extrusion, a mono-frequent sinusoidal signal and a bidirectional zeroing high-voltage pulse signal were selected as the controlling and electroporation signals, respectively. Based on this study, electric circuits for producing signal, intelligent control, display, and high-voltage power supply were designed, and corresponding software was also developed. At the same time, PCB was designed, electronic components were assembled and debugged, and parameters test was implemented. Finally, a cell-electrofusion instrument was manufactured, which can provide the electronic signal required for high-throughput fusion, and its technical parameters reached the international advanced level.4. Experiment study of cell electrofusion. The chip and cell-electrofusion instrument were integrated to form an experimental platform, and series of cell alignment and fusion experiments for different cells including microbiologic cells (yeast), animal cells (HEK-293 and chicken red blood cells), and plant cells (tobacco mesophyll protoplasts) were carried out. The result indicated that the chip with a great number of microelectrode (magnitude of 103 couples /cm2) could align and perforate lots of cells simultaneously, and the target of high-throughput cell electrofusion was also achieved. On the other hand, low voltage was required for cell manipulation and fusion for short distance between microelectrode pairs. Fax example, the peak-peak voltage (Vpp) for cell alignment is about 6 V, and the pulse voltage for cell electroporation is also less than 60 V. Lower voltage means a safer cell-electrofusion system, lower design requirements and production cost. Using a series of analyses, simulation and experimental study, microelectrode design, material choice and packaging technology were optimized, and it can generate a favorite electric field and electric-strength gradient distribution for cell-electrofusion. The experimental result indicated that a higher fusion rate could be obtained on the chip. For example, for the tobacco mesophyll protoplast, its fusion rate was as high as 50.2%. Furthermore, the cell-fusion rate reached a high level of 39.1% for HEK-293, 33.4% for yeast cells, respectively. Compared with traditional chemical fusion (polyethylene glycol PEG) (< 1%) and electrofuion method (< 10%), the fusion rate on our devices is much higher.In a word, based on the study of the electrode design and electric property, a high-throughout and high fusion-rate chip was developed. Furthermore, according to electric signal required for electrofusion, an intellectualized cell-fusion instrument was also set up. The instrument can be integrated with the chip to for an excellent experimental platform. On this platform, good experimental effect has been obtained in the cell fusion of microbiologic cells, animal cells, and plant protoplasts. This study made it possible to miniaturize cell-electrofusion system and set up a solid foundation for further research and development of an automatic cell-fusion chip laboratory.