Vadeikienė Roberta, Jakštys Baltramiejus, Ugenskienė Rasa, Šatkauskas Saulius, Juozaitytė Elona
Oncology Research Laboratory, Institute of Oncology, Lithuanian University of Health Sciences, LT-50161 Kaunas, Lithuania.
Research on Delivery of Medicine and Genes Cluster, Faculty of Natural Sciences, Vytautas Magnus University, LT-44001 Kaunas, Lithuania.
Biomedicines. 2022 Oct 24;10(11):2687. doi: 10.3390/biomedicines10112687.
Non-adherent cells are difficult to transfect with chemical-mediated delivery methods. Electroporation is an attractive strategy to transfer the molecules of interest into suspension cells. Care must be taken with the viability of the transfected cells since parameters, which increase cell membrane permeability, subsequently increase transfection efficiency, leading to higher cell death indices. We intended to evaluate the distribution of hard-to-transfect UT-7 cells among different subpopulations: transfected/viable, untransfected/viable, transfected/dead, and untransfected/dead populations, for a better understanding of the relation between gene electrotransfer efficacy and cell death. The following electroporation parameters were tested: pulse strength, duration, plasmid DNA concentration, and ZnSO as DNase inhibitor. BTX T820 square-wave generator was used, and 48 h after electroporation, cells were observed for viability and fluorescence analysis. Increasing pulse strength correlated directly with an increased ratio of pEGFP-positive cells and inversely with cell viability. The best results, representing 21% pEGFP positive/viable cells, were obtained after EP with 1 HV 1400 V/cm pulse of 250 µs duration using 200 µg/mL plasmid concentration. Results demonstrated that plasmid concentration played the most significant role in pEGFP electrotransfer into UT-7 cells. These results can represent a relevant improvement of gene electrotransfer to obtain genetically modified suspension cells for further downstream experiments.
非贴壁细胞很难通过化学介导的递送方法进行转染。电穿孔是一种将感兴趣的分子导入悬浮细胞的有吸引力的策略。由于增加细胞膜通透性的参数随后会提高转染效率，从而导致更高的细胞死亡指数，因此必须注意转染细胞的活力。我们旨在评估难以转染的UT-7细胞在不同亚群中的分布：转染/存活、未转染/存活、转染/死亡和未转染/死亡群体，以便更好地理解基因电转染效率与细胞死亡之间的关系。测试了以下电穿孔参数：脉冲强度、持续时间、质粒DNA浓度以及作为DNA酶抑制剂的硫酸锌。使用BTX T820方波发生器，电穿孔48小时后，观察细胞的活力并进行荧光分析。脉冲强度的增加与pEGFP阳性细胞比例的增加直接相关，与细胞活力呈负相关。在使用200μg/mL质粒浓度、1400V/cm的1HV 250μs脉冲进行电穿孔后，获得了最佳结果，即21%的pEGFP阳性/存活细胞。结果表明，质粒浓度在pEGFP向UT-7细胞的电转染中起最重要作用。这些结果可代表基因电转染的相关改进，以获得用于进一步下游实验的基因修饰悬浮细胞。
