基于薄膜电极的细胞电融合芯片

构建了一种薄膜电极阵列结构的细胞电融合芯片, 通过多聚物微通道底/顶层凸齿状的微电极, 以及多聚物微通道侧壁上溅射形成的一层离散式金属薄膜电极, 共同形成离散式"三明治"微电极结构. 该微电极结构可在微通道内部形成与传统凸齿状电极相似的非均匀分布的梯度电场, 通过介电电泳效应进行细胞控制及排队. 利用多聚物在芯片上填充了传统凸齿状电极的凹陷区, 克服了细胞在凹陷区无法有效排队与融合的缺点. 在芯片上利用K562细胞开展了基于介电电泳效应的细胞排队实验及基于可逆性电穿孔效应的电融合实验, 结果表明该芯片能够较好地实现细胞排队及融合, 融合所需控制电压低至10 V左右. 细胞排队率达99%以上, 几乎无细胞在绝缘物填充区(传统凸齿电极芯片的凹陷区)滞留, 细胞两两排队高于60%, 细胞融合效率约为40%, 比传统的细胞电融合方法和凸齿电极芯片有较大提高.     

高效细胞电融合芯片中的电场分析

细胞电融合芯片内的电场分布对细胞的控制及细胞融合效率有非常重要的意义,它是该类芯片设计的主要因素。电场分布主要由芯片内微通道和微电极的结构决定。在一个新研制的融合芯片中,采用大量微电极构成的阵列来提高融合效率。由于电极数量很多,微通道和微电极的结构和形状复杂,理论计算芯片内部电场分布具有较大难度。利用ANSYS有限元分析软件,对细胞电融合芯片中的电场分布进行模拟分析,得到其强度分布及变化梯度。通过不同设计的对比分析,提出了更加适合于细胞电融合的电极阵列结构模型——矩形梳状交叉微电极阵列,为高效细胞电融合芯片的实现奠定了基础。在矩形梳状交叉微电极阵列原型芯片的实验研究中,细胞融合(植物原生质体融合)效率约为40%,超过了传统的化学融合(小于1%)、电融合(小于10%),以及最初所采用的矩形对称梳状电极(小于20%)。表明在该融合芯片上可以实现高效的细胞电融合。     

基于聚酰亚胺基底的细胞电融合芯片的研制

基于柔性印刷电路板(flexible printed circuits board, FPCB)技术,通过在聚酰亚胺基底薄膜表面层压的铜箔上刻蚀微电极阵列结构制备了一种细胞电融合芯片.在低电压(≤40 V)条件下实现了细胞电融合,融合效率达37%,远高于聚乙二醇(polyethylene glycol, PEG)法及传统细胞电融合方法.与传统细胞电融合系统相比,此芯片可在低电压条件下工作,具有结构简单、成本低廉、实验过程可观察、融合通量高等优点.另外,聚酰亚胺薄膜基底良好的柔软度可保证此芯片与其它分析模块(如细胞筛选分离模块)的有效集成,具备构造微全分析系统(micro total analytical system, μ-TAS)的巨大潜力.     

微电极阵列芯片的脂质体电融合研究

利用旋转蒸发法制作基于大豆卵磷脂的一种大型脂质体,在微电极阵列芯片上进行脂质体电融合实验研究.在电融合过程中,利用介电电泳力实现脂质体在微流控芯片中的排队,再利用高场强的电脉冲使脂质体膜发生可逆性电穿孔,在持续的介电电泳力作用下,使穿孔的脂质体实现融合.芯片上脂质体的融合率可以达到20％左右.而且,玻璃基底材料和低深宽比的通道结构更有利于脂质体融合过程的观察与控制.     

基于微流体脉冲驱动控制技术的电化学微流控芯片制备方法

基于微流体脉冲驱动控制技术搭建了电化学微流控芯片的制备系统.首先将纳米银墨水和甘油溶液分别微喷射到玻璃基底表面形成微电极图形和微流道液体阳模图形;然后分别进行烧结和聚二甲基硅氧烷(PDMS)模塑工艺制得微电极和微流道;最后将微电极和微流道键合形成电化学微流控芯片.研究了系统参量对液滴产生的影响以及液滴直径和重叠率对液滴成线的影响,制得的微电极最小线宽为45 μm、厚度为2.2 μm、电阻率为5.2 μΩ·cm,制得的微流道最小线宽为35 μm,流道表面光滑.采用制得的电化学微流控芯片进行了葡萄糖浓度的电化学流动检测.结果表明,葡萄糖溶液的浓度与响应电流具有较高的线性关系,可对一定浓度范围内的葡萄糖溶液进行定量检测.基于微流体脉冲驱动控制技术的电化学微流控芯片制备方法具有微喷射精度高、重复性好,制备系统结构简单、成本低廉等优点,可用于生化分析、生物传感器等领域的芯片制备.     

用于细胞排列的介电泳微流控芯片制备与实验研究

设计并制作了一种应用于细胞排列的介电泳微流控芯片,以实现细胞的非接触、批量排列。芯片主要包括PDMS微通道和“台阶”形ITO微电极。运用仿真软件COMSOL分析了微电极所形成的电场分布,确定了最大电场强度的位置;利用MEMS加工工艺制备了ITO微电极和PDMS微通道,PDMS微通道与带有ITO电极的载玻片经过氧等离子表面处理后,对准键合获得最终的微流控芯片。通过不同频率下的介电泳实验,实现了酵母菌细胞的介电泳运动,并确定了正、负介电泳运动的电场频率。结果表明,酵母菌细胞在溶液电导率为60μS/cm的环境下,1~10 kHz时,发生负介电泳运动;0.5~10 MHz时,发生正介电泳运动;50 kHz时,没有发生介电泳运动。并在施加8 Vp-p,5 MHz交流电压信号的条件下,实现了酵母菌细胞沿“台阶”形电极边缘直线排列。     

IBRS2细胞电融合及融合过程研究

单层贴壁培养的IBRS2细胞在高压脉冲电场的诱导下,能发生大规模的融合。当脉冲强度达1.7kV/cm,脉冲宽度达100μs,脉冲个数达4,电击液中含lmol/LMg~(2+)时,融合率可达90％以上。电击液中钙、镁离子的浓度对细胞的电融合亦有明显的影响。实验表明,钙离子能抑制IBRS 2细胞的电融合并降低细胞对电脉冲的耐受能力。反之,镁离子则能促进IBRS 2细胞的电融合并提高细胞对电脉冲的耐受能力。仔细的实验观察表明,IBRS 2细胞的电融合是一个十分复杂、缓慢的动态过程。依据实验结果,我们对该种细胞的电融合过程和机制提出了一种可能的解释。     

细胞电融合芯片技术的发展及展望

细胞电融合芯片技术是最近十几年来发展迅速的一种细胞融合方法,它可以广泛用于遗传学、动植物远缘杂交育种、发育生物学、免疫学、医药、食品以及农业等领域的基础研究和应用开发。由于其不仅具有可控性强、操作简便、对细胞无毒害等优点,还比传统细胞电融合技术更安全、快捷、高效、便携、集成度高,而且便于实验观察,样本消耗少,因此,在国内外广受关注。本文综述了细胞电融合芯片技术的基本原理、实现方法、研究进展,并对其未来发展作了展望。     

小鼠2-细胞胚胎电融合的研究

本试验对影响小鼠2-细胞胚胎电融合诸因素进行了研究。胚胎细胞融合面与两电极相平行或相垂直;胚胎细胞在0.3 mol/L甘露醇液、0.25mol/L蔗糖液和杜氏磷酸缓冲液(PBS)中;当电脉冲的持续时间为80μs,电场强度为0,800,1000,1200,1400和1600V/cm时以及当电脉冲的电场强度为1200V/cm,持续时间为10,20,40,80,160和320μs时,胚胎细胞融合效果的比较结果表明:融合面与两电极相平行,以0.3mol/L甘露醇液为融合液,电脉冲在1000—1200V/cm,20—160μs范围内的条件下,可获得满意的胚胎细胞融合率(88.6—94.5％),平均为92.2％。     

葡聚糖及其衍生物对大麦细胞电融合的影响

葡聚糖及其衍生物对大麦细胞电融合的影响①王伟江志裕＊（复旦大学化学系上海２００４３３）细胞融合是指用人工方法使两个细胞融合成一个细胞的过程［１,２］．体细胞融合可在不同种,不同属,甚至更远的生物间进行,所得到的细胞可具有新的特性．对于植物细胞,有望通...     

A high-throughput dielectrophoresis-based cell electrofusion microfluidic device

A high-throughput cell electrofusion microfluidic chip has been designed, fabricated on a silicon-on-insulator wafer and tested for in vitro cell fusion under a low applied voltage. The developed chip consists of six individual straight microchannels with a 40-μm thickness conductive highly doped Si layer as the microchannel wall. In each microchannel, there are 75 pairs of counter protruding microelectrodes, between which the cell electrofusion is performed. The entire highly doped Si layer is covered by a 2-μm thickness aluminum film to maintain a consistent electric field between different protruding microelectrode pairs. A 150-nm thickness SiO? film is subsequently deposited on the top face of each protruding microelectrode for better biocompatibility. Owing to the short distance between two counter protruding microelectrodes, a high electric field can be generated for cell electrofusion with a low voltage imposed across the electrodes. Both mammalian cells and plant protoplasts were used to test the cell electrofusion. About 42-68% cells were aligned to form cell-cell pairs by the dielectrophoretic force. After cell alignment, cell pairs were fused to form hybrid cells under the control of cell electroporation and electrofusion signals. The averaged fusion efficiency in the paired cells is above 40% (the highest was about 60%), which is much higher than the traditional polyethylene glycol method (<5%) and traditional electrofusion methods (～12%). An individual cell electrofusion process could be completed within 10 min, indicating a capability of high throughput.     

A cell electrofusion microfluidic chip using discrete coplanar vertical sidewall microelectrodes

A novel cell electrofusion microfluidic chip using discrete coplanar vertical sidewall electrodes has been designed, fabricated, and tested. The device contains a serpentine-shaped microchannel with 22 500 pairs of vertical sidewall microelectrodes patterned on two opposing vertical sidewalls of the microchannel. The adjacent microelectrodes on each sidewall are separated by coplanar SiO(2) -Polysilicon-SiO(2) /silicon. This design of coplanar discrete vertical sidewall electrodes eliminates the "dead area" present in previous designs using continuous three-dimensional (3D) protruding sidewall electrodes, and generates uniform electric field along the height of the microchannel, leading to a lower voltage required for cell fusion compared to designs using 2D thin-film electrodes. This device is tested to fuse NIH3T3 cells under a low voltage (～9 V). Almost 100% cells are aligned to the edge of the discrete microelectrodes, and cell-cell pairing efficiency reaches 70%. The electrofusion efficiency is above 40% of the total cells loaded into the device, which is much higher than traditional fusion methods and existing microfluidic devices using continuous 3D protruding sidewall microelectrodes.     

On chip electrofusion of single human B cells and mouse myeloma cells for efficient hybridoma generation

This article describes the development and full characterization of a microfluidic chip for electrofusion of human peripheral blood B-cells and mouse myeloma (NS-1) cells to generate hybridomas. The chip consists of an array of 783 traps, with dimensions that were optimized to obtain a final cell pairing efficiency of 33±6%. B cells were stained with a cytoplasmic stain CFDA to assess the different stages of cell fusion, i.e. dye transfer to NS-1 cells (initiating fusion) and membrane reorganization (advanced fusion). Six DC pulses of 100 μs (2.5 kV/cm) combined with an AC field (30 s, 2 MHz, 500 V/cm) and pronase treatment resulted in the highest electrofusion efficiency of paired cells (51±11%). Hybridoma formation, with a yield of 0.33 and 1.2%, was observed after culturing the fused cells for 14 days in conditioned medium. This work provides valuable leads to improve the current electrofusion protocols for the production of human antibodies for diagnostic and therapeutic applications.     

Optimization of bulk cell electrofusion in vitro for production of human-mouse heterohybridoma cells

Cell electrofusion is a phenomenon that occurs, when cells are in close contact and exposed to short high-voltage electric pulses. The consequence of exposure to pulses is transient and nonselective permeabilization of cell membranes. Cell electrofusion and permeabilization depend on the values of electric field parameters including amplitude, duration and number of electric pulses and direction of the electric field. In our study, we first investigated the influence of the direction of the electric field on cell fusion in two cell lines. In both cell lines, applications of pulses in two directions perpendicular to each other were the most successful. Cell electrofusion was finally used for production of human-mouse heterohybridoma cells with modified Koehler and Milstein hybridoma technology, which was not done previously. The results, obtained by cell electrofusion, are comparable to usually used polyethylene glycol mediated fusion on the same type of cells.     

Electro‐optical characteristics in phase separated liquid crystal/photo‐curable acrylic monomer mixture system

One of the main objectives of the experiment was to achieve the vertical aligned (VA) effect. To accomplish this, we employed liquid crystal (LC)/photo‐curable acrylic monomers mixture systems to prepare vertical alignment copolymer film (VACOF) for LC molecules with the photo‐polymerization induce phase separation (PIPS) process. From previous experimental results, we successfully fabricated LC devices without the micro‐protrusion structure. After the application of a saturated voltage, the LC molecules actually exhibited such interesting phenomena as uniaxial orientation, uniform single‐domain display state, etc. In this study, to obtain VACOF with smooth surface, we similarly controlled appropriate experimental conditions such as UV light exposure intensity and curing temperature, and altered process parameters such as the cell thickness, chemical structure length of the main chain type biphenol acrylic monomer [to simulate the main chain function of the traditional vertical alignment type polyimide (PI)], etc. During the experiment, we discovered that regardless of the cell thickness, this photo‐alignment system would yield the VACOF instead of the polymer disperse liquid crystal (PDLC) film morphology. Another notable finding was that the contrast ratio was heavily influenced by the length of the main chain type acrylic monomer structure for LC/monomer mixture systems, with enhancement of up to ～56%. Therefore, we further investigated the display effects, electro‐optical properties, etc. for these two main chain type acrylic monomer systems with different lengths and cell thicknesses. Copyright © 2011 John Wiley & Sons, Ltd.     

Effects of off-state alignment in polymer dispersed liquid crystals

Partial off-state alignment of the liquid crystal in polymer dispersed liquid crystal (PDLC) droplets was obtained by the application of electric or magnetic fields during their formation. Photopolymerization was used to induce phase separation of the liquid droplets from monomer/liquid crystal solutions. Substantial director directionality was retained in these PDLC films after removal of the fields used during their formation. This alignment affected both the off-state and the on-state electro-optic properties of the films. Transverse electrical fields (5 to 60 V across a 15 μm thickness) applied during PDLC formation from a solution of E7 (BDH Ltd) in a monomer resulted in PDLC films with progressively lower off-state scattering and lower threshold voltage. Strong longitudinal magnetic fields (9 to 14 T) applied during PDLC formation with these materials resulted in strong polarization effects in the light scattering off-state. In the infrared region, where there is less light scattering than in the visible region, the longitudinally aligned films shows tunable birefringent electro-optic effects while retaining the fast time response characteristics of PDLC films with small droplet sizes.     

Dielectrophoresis-assisted massively parallel cell pairing and fusion based on field constriction created by a micro-orifice array sheet

In this paper, we present a novel electrofusion device that enables massive parallelism, using an electrically insulating sheet having a two-dimensional micro-orifice array. The sheet is sandwiched by a pair of micro-chambers with immersed electrodes, and each chamber is filled with the suspensions of the two types of cells to be fused. Dielectrophoresis, assisted by sedimentation, is used to position the cells in the upper chamber down onto the orifices, then the device is flipped over to position the cells on the other side, so that cell pairs making contact in the orifice are formed. When a pulse voltage is applied to the electrodes, most voltage drop occurs around the orifice and impressed on the cell membrane in the orifice. This makes possible the application of size-independent voltage to fuse two cells in contact at all orifices exclusively in 1:1 manner. In the experiment, cytoplasm of one of the cells is stained with a fluorescence dye, and the transfer of the fluorescence to the other cell is used as the indication of fusion events. The two-dimensional orifice arrangement at the pitch of 50 μm realizes simultaneous fusion of 6 × 103 cells on a 4?mm diameter chip, and the fusion yield of 78-90% is achieved for various sizes and types of cells.