摘要
细胞电融合技术自从由Zimmermann发明以来,就因为效率高、可控性强、操作简便、重复性好、对细胞无毒害等优点而得到广泛应用,逐渐成为现代生物工程技术中的一个重要工具,被广泛应用于物种产生或改良、单克隆抗体制备、癌症免疫治疗等领域。
本文运用现代微机电系统(micro electro mechanical systems, MEMS)加工技术在厘米见方的基底材料上制作了微电极阵列细胞电融合芯片。所包含的微电极对数达到103数量级,微电极间距小于100μm。同时,研究开发了细胞电融合仪,可以与芯片集成为高通量细胞电融合系统,并利用介电电泳和细胞电致穿孔原理实现细胞操控和电融合。
细胞定位、穿孔及挤压操作是细胞电融合的基础。因此,本文首先对细胞操控和电融合原理进行了深入的探讨,在此基础上运用有限元方法分析了不同形状、分布的微电极阵列在外加电压条件下的电场分布,并以此为据来优化微电极和微电极阵列设计,以获得有利于细胞电融合的电场环境。在此期间确定了矩形梳状交叉微电极阵列芯片模型。随后,对芯片加工工艺、材料、封装技术等进行了研究,成功研制了硅基底-硅微电极、玻璃基底-硅微电极、硅基底-全金属微电极、电路板(printed circuit board, PCB)微电极等四种不同材料的细胞电融合芯片。同时,研制开发了细胞电融合仪。最后,在芯片和融合仪组成的高通量细胞电融合平台上进行了微生物、动物、植物细胞电融合实验。具体来说,论文研究工作主要包括以下几个方面:
1.芯片上微电极尺寸及分布对电场强度及梯度的影响。在细胞融合芯片上,三维微电极的几何及分布参数包括微电极厚度、宽度、深度、纵向间隔、横向间隔,微电极尖端几何形状(直角形齿、锥形齿、平行板),电极排列方式(对称性和非对称型)等。这些参数的改变都会对微通道中的电场强度及梯度产生影响。通过有限元方法,对微电极阵列进行了建模仿真。根据细胞电融合的要求,提出了一种有利于细胞电融合的矩形梳状交叉微电极阵列芯片模型,为实现高效细胞电融合奠定了理论基础。在该芯片中,可以产生有利于细胞电融合的电场环境,提高对细胞的操控能力和融合率。
2.芯片和微电极材料及制作工艺的选择。为了使细胞电融合芯片获得最佳的电气和生化性能,本文研究了全硅微电极、硅玻结合(玻璃基底加硅微电极)、硅基底全金属微电极、PCB铜电极等四种芯片,包括其微电极和微通道设计、加工工艺选择、封装设计和材料选择等。
3.细胞电融合仪的设计及制作。在研究了细胞电融合过程中涉及到的细胞操作,即排队、电穿孔、挤压等三个基本过程及所需电信号特征后,提出了用单频正弦信号和双向归零高压脉冲信号作为细胞电融合的最佳操控与穿孔信号。在此基础上,设计了信号产生电路、智能控制电路、显示电路、高压电源电路并进行相应的软件编写、PCB设计、电子装配与调试,参数测试等,获得了可以提供高通量细胞电融合所需电信号的细胞电融合仪,其技术指标达到国内先进水平。
4.细胞电融合实验研究。利用新研制的芯片和融合仪建立了细胞电融合实验平台,开展了微生物(酵母细胞)、动物(HEK-293、鸡血细胞)、植物(烟草叶肉原生质体)细胞的排队或融合实验。结果表明,芯片内数量巨大微电极(103对/cm2数量级)可以同时操控大量细胞来完成排队和电穿孔,从而实现高通量电融合。另一方面,由于微电极间距小到微米数量级,细胞操作和融合所需电压很低,如细胞排队电压(Vpp)在6 V左右即可达到满意效果,电穿孔脉冲电压幅值也小于60 V。这样,融合电压的降低提高了细胞电融合系统安全性,也降低了电融合仪的设计要求和生产成本。通过一系列分析、仿真及实验研究,使芯片内微电极的设计、选材及加工工艺得以优化,可以产生更有利于细胞电融合的电场和电场梯度分布。实验结果表明,芯片上的细胞电融合可以获得较高的电融合率。以烟草叶肉原生质体的电融合为例,其融合率最高达到50.2%。动物细胞HEK-293的融合率最高也达39.1%,酵母细胞最高达33.4%,这些都较传统的利用聚乙二醇(polyethylene glycol, PEG)的化学融合方法的融合率(< 1%)和传统电融合仪器上的融合率(< 10%)高出很多。
总之,通过对细胞电融合芯片电极设计以及电学特性的研究,研制出高通量、高融合率的细胞电融合芯片,并根据细胞电融合过程和所需电信号要求,研制开发出智能化细胞电融合仪。集成融合芯片及电融合仪建立了细胞电融合微系统实验平台,在对微生物、动物、植物原生质体等细胞的电融合实验中,取得了很好的实验效果。该研究为实现细胞电融合系统微型化、建立高效、自动的细胞融合芯片实验室奠定了良好的基础。
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.
引文
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