Surviving high-intensity field pulses:: Strategies for improving robustness and performance of electrotransfection and electrofusion
Surviving high-intensity field pulses:: Strategies for improving robustness and performance of electrotransfection and electrofusion
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DOI：
10.1007/s00232-005-0791-2
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发表时间：
2005-08-01
影响因子：
2.4
通讯作者：
Zimmermann, U
中科院分区：
文献类型：
--
作者：
Sukhorukov, VL;Reuss, R;Zimmermann, U
电转染和电融合广泛应用于研究和医学应用，但仍然面临一系列问题，包括电穿孔抗性细胞类型的存在、细胞死亡率以及转染和融合产量的批次间巨大差异。在本研究中，对五种哺乳动物细胞类型对电操作方案的效率和稳健性至关重要的参数进行了系统分析。检查的因素包括低渗脉冲培养基的糖成分（海藻糖、山梨糖醇或肌醇）、电脉冲前细胞体积变化的动力学，以及用于脉冲后细胞培养的生长培养基添加剂。二糖海藻糖通常会导致调节体积减少 (RVD)，而单体糖醇山梨醇和肌醇会抑制 RVD，甚至诱导继发性肿胀。不同的体积反应可以通过所有测试细胞类型质膜中体积敏感通道（VSC）的糖选择性来解释。根据体积数据，当靶细胞在电脉冲前暴露于低渗约 2 分钟时，大多数转染和融合产量都达到最高。较长的低渗处理（10-20 分钟）会降低活转染细胞和杂交细胞的产量，原因是（1）RVD（海藻糖）时细胞尺寸减小或（2）VSC（肌醇/山梨醇）导致胞质电解质过度损失。用亲脂性阴离子掺杂质膜可防止细胞收缩和离子损失（可能是由于 VSC 抑制），从而提高转染和融合效率。
Electrotransfection and electrofusion, both widely used in research and medical applications, still have to face a range of problems, including the existence of electroporation-resistant cell types, cell mortality and also great batch-to-batch variations of the transfection and fusion yields. In the present study, a systematic analysis of the parameters critical for the efficiency and robustness of electromanipulation protocols was performed on five mammalian cell types. Factors examined included the sugar composition of hypotonic pulse media (trehalose, sorbitol or inositol), the kinetics of cell volume changes prior to electropulsing, as well as the growth medium additives used for post-pulse cell cultivation. Whereas the disaccharide trehalose generally allowed regulatory volume decrease (RVD), the monomeric sugar alcohols sorbitol and inositol inhibited RVD or even induced secondary swelling. The different volume responses could be explained by the sugar selectivity of volume-sensitive channels (VSC) in the plasma membrane of all tested cell types. Based on the volumetric data, highest transfection and fusion yields were mostly achieved when the target cells were exposed to hypotonicity for about 2 min prior to electropulsing. Longer hypotonic treatment (10-20 min) decreased the yields of viable transfected and hybrid cells due to (1) the cell size reduction upon RVD (trehalose) or (2) the excessive losses of cytosolic electrolytes through VSC (inositol/sorbitol). Doping the plasma membrane with lipophilic anions prevented both cell shrinkage and ion losses (probably due to VSC inhibition), which in turn resulted in increased transfection and fusion efficiencies.