High-throughput Cell Electrofusion Chip And Experimental Research

Cell electrofusion technology is applying electric stimulation to let two or more cells merge into a hybrid cell in an asexual way. In comparison with the traditional biological, chemical and physical mediated cell fusion method, cell electrofusion technique has many advantages such as high efficiency, easy operation, no contamination and visual fusion process. It has becoming an important tool in biological technology and bioengineering research, and been widespread used in genetics, animal and plant hybridization, biological evolution, immunology, drug development, food industry. Especially, it has become a key technology in crossbreeding, drug screening, antibody production, cancer vaccine development.MEMS technology helps the research of cell electrofusion go deep into microscale. Microfabrication can achieve micrometer scale structure and integrate a large number of microelectrodes on a chip to realize makes high-throughput cell electrofusion. Moreover, micrometer distance between microelectrode pair decreases the requirement of voltage during the fusion. Thus, cell electrofusion chip can work in low-voltage condition.Commercial cell electrofusion systems have some problem, like low efficiency in cell alignment and cell electrofusion. Our group has developped a cell electrofusion chip to solve these problems. It has maded some progress in cell alignment (the cell alignment efficiency up to 69.2±18.5%). And its fusion efficiency in paired cells (50.2%) was much larger than those in traditional chemical induced fusion (1‰) and electrofusion (1%). But for high-throughput cell electrofusion chip promote the use of engineering there is a large distance. Especailly the efficiency of cell alignment and cell electrofusion need to improve. In the same time, the research on operability, cluture of fused cell, and the fused cell karyotyping are still exist. Therefore, combined with these problems, this paper carried out research as follws:In this paper, based on the research on the classic theory of cell dielectrophoresis and cell electroporation, the profile of electric field on the chip was analyzed by using finite element method and the COMSOL simulation software. The researching emphasis was focused on those factors which could impact the profile of the electric field and the simulation results were used to guide the optimization of chip structures. High-efficiency cell-alignment control and electrofusion method have been established on the theory analysis and simulation. It aims to improve the efficiency of chip in cell alignment and cell electrofusion. Combined with micro-processing technology, a series of cell-electrofusion chip based on SOI wafer, flexible polymer (PI substrate), silicon-glass bonding wafer, and surface-electrode technique were developed.1. Theoretical research and simulation of electric field profile on the chipBased on classical electromagnetic, dielectrophoretic, cell-electrofusion theories, the electric-field profile on the cell-electrofusion chip was analyzed by using finite element method and COMSOL simulation software. In particular, the relationship between electric field intensity, electric field gradient and the structure parameters such as arrangement and size of the microelectrodes was computed. According to the simulation results, optimized microelectrode structures were developed and high-efficiency cell alignment control and electrofusion method was established.2. A series of cell-electrofusion chipBased on theoretical analysis and simulation, a series of cell-electrofusion chip were developed. Comprehensively thinking of the reliability, stability, integration, biocompatibility, cell aligment, fusion efficiency, materials, fabrication technique, package and cost, optimized chip design and a series of cell-electrofusion chips were developed.3. Establishment of the cell-electrofusion platformAccording to the specific requirements of the experimental research and the characteristics of the series cell electrofusion chip mentioned above, two cell electrofusion platforms were established.4. Cell-electrofusion experimental researchOn the cell electrofusion platform, many cell-electrofusion experiments based on animal, plant, microorganism cells were carried out. Experimental results showed that these chips could work under low-voltage conditions, and the cell alignment voltage was only 2~10 V and the cell-electrofusion voltage was 8~60 V. A great number of microelectrodes (103~105 on the chip) could be used to control lots of cells to realize cell alignment and electroporation. It was the base for high-throughput cell electrofusion. In the electrofusion experiments, high-efficiency cell alignment control has been realized. On the chip, more than 90±9.5% cells moved towards the microelectrodes and were aligned by the dielectrophoretic force which was generated by the alignment signal. By adjusting the electric-filed intensity, most cells could be controlled to align as cell-cell pairs (homologous cells cell-cell pairs rate of 40~60%). Based this high-efficiency cell alignment, cell pairs were fused to form hybrid cells under the control of cell electroprotaion signal. The fusion efficiency in pairs cells, more than 60% (the best level reached 90%), which was a great improvement compared with traditional PEG method (less than 1‰) and electrofusion methods (less than 1%).In cell-electrofusion experiments about microorganisms based on the SOI cell electrofusion chip, strains BS-34 was choosen as researching samples. The impact of the experiment parameters such as the ion concentration, voltage, etc. on the cell alignment and electrofusion was explored. The surface tension of fused strains increased from 62% to 85%, which showed that this cell-electrofusion system had great potential in strains promotion.In the electrofusion experiments on plant protoplasts based on the PI substrate cell electrofusion chip, tobacco protoplasts were used to constructed tetraploid tobacco hybrid. Relative experimental research has been carried out.In the electrofusion experiments on animal cells based on the silicon-glass bonding chip, the impact of many parameters on the fusion efficiency has been explored and relative fusion experiments have been carried out. For example, NIH3T3 cells and mMESc were used as samples to research the electrofusion between somatic cells and stem cells. These results validated that our system could perform high-efficiency cell electrofusion process. Culture and karyotype analysis of fused cells also proved the feasibility and effectiveness of this system. This chip could produce large amounts of fused cells at the same time and it established a good foundation for somatic cell reprogramming research.In conclusion, by using the theoretical and simulation research, the on-chip profile of electric field and other factors have been analyzed. A series of chip and electrofusion platform have been developed. Experimental research on animal, plant and microbial cells have been done, which set up good foundation for strains improvement, plant breeding, and reprogramming of somatic cells.Meanwhile, some design and research for future working have been explored, whose objectives were to realize miniaturization, integration and systematization, and set up a whole system integrated with sample preparation, on-chip electrofusion, detection and separation of fused cells, and on-chip cell culture.