Agents facilitating the electric field-induced fusion of intact rabbit erythrocytes

In the recent years, the role of specific membrane active agents in the electrofusion process has started to draw attention, and it has been found that the presence of various substances in the cell medium can affect the fusion process either in a positive or negative way. In this work, the effect of several proteins, bivalent cations and antibiotics was tested with respect to their ability to protect intact erythrocytes from hemolysis and facilitate the fusion process. The effect of different sugars was also studied. Among the different proteins, pronase and proteinase were found to be the most effective. With respect to bivalent cations, Ca2+ and Mn2+ were more effective while Mg2+ was less important. From the antibiotics, penicillin caused a negative effect while streptomycin acted positively. Finally, glucose medium was found to be the most effective compared to all sugars tested. The results indicated that there are strong differentiations of the induced effects caused by each substance, and some possible mechanisms of action of these agents on the erythrocyte membrane were discussed.     

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.     

用荧光葡聚糖研究大麦细胞电融合

利用阴离子表面活性物质荧光葡聚糖 (F DX)研究了表面活性物质对大麦细胞电融合的影响 .结果表明 ,F DX可抑制电融合过程 .对放置过大麦细胞原生质体的F DX溶液 ,在荧光显微镜下可观察到其膜表面的荧光圈 ,证明F DX在膜上的吸附 .添加F DX可增加原生质体的电泳速度 ,说明吸附后原生质体表面负电荷增多 .由于相互间静电斥力的增强 ,使细胞的电融合率下降 .此外 ,还利用荧光显微技术研究了细胞电生孔现象 .观察到经电脉冲后溶液中的F DX可进入原生质体内部 ,间接证明了细胞电生孔的存在 .     

Electrofusion of an industrial Baker’s yeast strain with a sour dough yeast

A method for hybridization of yeast protoplasts ofSaccharomyces cerevisiae andCandida holmii by electrofusion was optimized. The hybrids were screened on maltose-acetate agar plates. The average fusion frequency was 1.1xl0-³. Two hybrids of 132 collected from selection plates were found to be stable over 15 sequential shake flask cultivations. However, the strains reverted during production of baker’s yeast in a laboratory scale process imitating the industrial process.     

High-resolution analyses of cell fusion dynamics in a biochip

In this study, we analyzed the electrofusion of two cells in a biochip that has been developed to perform the capture by dielectrophoresis and the electrofusion of pairs of cells. The good transparency of the microsystem allowed analyzing the details of the fusion events. By staining one of the cells, the mixing of the two cytosols could be observed during the electrofusion experiment. We show for the first time the rapidity of the mixing of the two cytosols: less than 5 s under our experimental conditions. By comparing these experimental results to a numerical simulation, we found that the rate of this phenomenon is compatible with a diffusion-only mechanism, showing that during the fusion, the two cell membranes in contact are affected by very rapid structural changes and do not limit the exchange of the cytosols between the two cells. A point of interest is the use of dielectric structures to concentrate the electric field and of positive dielectrophoresis to capture cells in the area where the electric field is more intense. This technique allows the increase of the cell-to-cell contact and limits cell cytosol leakages during the fusion process.     

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。