Rybakovsky Elizabeth, Valenzano Mary Carmen, DiGuilio Katherine M, Buleza Nicole B, Moskalenko Daniil V, Harty Ronald N, Mullin James M
Lankenau Institute for Medical Research, Wynnewood, Pennsylvania 19096, USA.
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
J Biomol Tech. 2019 Jul;30(2):19-24. doi: 10.7171/jbt.19-3002-001.
Polar, differentiated epithelial cell culture models (especially at confluence) are difficult to transfect compared with the higher transfection efficiencies that one obtains with relatively less differentiated, nonpolar cell culture models. Here, we sought to develop a strategy to enhance the efficiency of transfecting polar, differentiated epithelial cells. We found that chemically abrading the differentiated CACO-2 human intestinal epithelial cell layer by a trypsin and EDTA pretreatment (before the use of detergent-like transfection reagents) dramatically improved transfection efficiency in this polar, differentiated model. Although this treatment did improve the transfection efficiency, it also induced leakiness in the epithelial barrier by both opening tight junctional complexes and by creating holes in the cell layer because of low-level cell death and detachment. Thus, this approach to enhance the transfection efficiency of polar, differentiated cells will be useful for assessment of the effect of the transfected/expressed protein on (re)formation of an epithelial barrier rather than on a functional barrier itself.
与相对未分化的非极性细胞培养模型相比，极性分化上皮细胞培养模型（尤其是汇合时）转染难度较大，后者能获得更高的转染效率。在此，我们试图开发一种策略来提高极性分化上皮细胞的转染效率。我们发现，在用胰蛋白酶和乙二胺四乙酸预处理（在使用类似去污剂的转染试剂之前）对分化的Caco-2人肠上皮细胞层进行化学磨损，可显著提高该极性分化模型中的转染效率。尽管这种处理确实提高了转染效率，但由于低水平的细胞死亡和脱离，它还通过打开紧密连接复合物和在细胞层中形成孔洞，导致上皮屏障渗漏。因此，这种提高极性分化细胞转染效率的方法将有助于评估转染/表达的蛋白质对上皮屏障（再）形成的影响，而不是对功能性屏障本身的影响。
