Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia.
Electrophoresis. 2012 Sep;33(18):2867-74. doi: 10.1002/elps.201200265.
We describe the development and testing of a setup that allows for DEP field-flow fractionation (DEP-FFF) of irreversibly electroporated, reversibly electroporated, and nonelectroporated cells based on their different polarizabilities. We first optimized the channel and electrode dimensions, flow rate, and electric field parameters for efficient DEP-FFF separation of moderately heat-treated CHO cells (50°C for 15 min) from untreated ones, with the former used as a uniform and stable model of electroporated cells. We then used CHO cells exposed to electric field pulses with amplitudes from 1200 to 2800 V/cm, yielding six groups containing various fractions of nonporated, reversibly porated, and irreversibly porated cells, testing their fractionation in the chamber. DEP-FFF at 65 kHz resulted in distinctive flow rates for nonporated and each of the porated cell groups. At lower frequencies, the efficiency of fractionation deteriorated, while at higher frequencies the separation of individual elution profiles was further improved, but at the cost of cell flow rate slowdown in all the cell groups, implying undesired transition from negative into positive DEP, where the cells are pulled toward the electrodes. Our results demonstrate that fractionation of irreversibly electroporated, reversibly electroporated, and nonelectroporated cells is feasible at a properly selected frequency.
我们描述了一种设置的开发和测试，该设置允许基于其不同的极化率对不可逆电穿孔、可逆电穿孔和未电穿孔的细胞进行 DEP 场流分级（DEP-FFF）。我们首先优化了通道和电极尺寸、流速和电场参数，以有效分离经适度热处理（50°C 15 分钟）的 CHO 细胞和未经处理的 CHO 细胞，前者用作电穿孔细胞的均匀且稳定的模型。然后，我们使用幅度为 1200 至 2800 V/cm 的电场脉冲处理 CHO 细胞，得到包含各种非穿孔、可逆穿孔和不可逆穿孔细胞分数的六个组，在腔室内测试它们的分级。在 65 kHz 时，DEP-FFF 导致非穿孔和每个穿孔细胞组的独特流速。在较低频率下，分级效率恶化，而在较高频率下，各个洗脱谱的分离进一步改善，但所有细胞组的细胞流速都减慢，这意味着从负 DEP 向正 DEP 的不期望转变，其中细胞被拉向电极。我们的结果表明，在适当选择的频率下，不可逆电穿孔、可逆电穿孔和未电穿孔的细胞的分级是可行的。
