Improving Transient Transfection Efficiency in a Differentiated, Polar Epithelial Cell Layer.

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.

Spontaneous and cytokine-induced hole formation in epithelial cell layers: Implications for barrier function studies with the gingival cell culture, Gie-3B11, and other epithelial models.

Epithelial barrier function studies often attribute alterations in barrier function to induced changes in tight junctional (TJ) complexes. The occurrence of spontaneous and cytokine-induced, focal cell detachment in cell layers of the human gingival epithelial cell line, Gie-3B11, highlights the danger of this assumption without confirmatory experimentation. Gie-3B11 cell layers manifest morphological polarity, TJ complexes and barrier function after confluence but fail to then maintain a stable epithelial barrier. Transepithelial electrical resistance rises to over 100 ohms x cm2 a few days after seeding cell layers at a confluent density, but then spontaneously declines, with simultaneous, inverse changes in transepithelial 14C-D-mannitol diffusion rates. This barrier decline correlates with the appearance of focal cell detachment/hole formation in cell layers. Both barrier compromise (decreased electrical resistance; increased 14C-D-mannitol leak) and hole formation are accelerated and exaggerated by exposing cell layers to proinflammatory cytokines. Both are inhibited by increasing the basal-lateral medium compartment volume, suggesting that cell layers are secreting factor(s) across their basal-lateral surfaces that are causal to hole formation. The molecular mechanism of cell death/detachment here is not as significant as the implications of hole formation for the correct interpretation of barrier function studies. Barrier changes in any epithelial model should be attributed to induced changes in TJ complexes only after thorough investigation.