Cell-cell proximity effects in multi-cell electroporation.

We report a fundamental study of how the electropermeabilization of a cell is affected by nearby cells. Previous researchers studying electroporation of dense suspensions of cells have observed, both theoretically and experimentally, that such samples cannot be treated simply as collections of independent cells. However, the complexity of those systems makes quantitative modeling difficult. We studied the change in the minimum applied electric field, the threshold field, required to affect electropermeabilization of a cell due to the presence of a second cell. Experimentally, we used optical tweezers to accurately position two cells in a custom fluidic electroporation device and measured the threshold field for electropermeabilization. We also captured video of the process. In parallel, finite element simulations of the electrostatic potential distributions in our systems were generated using the 3-layer model and the contact resistance methods. Reasonably good agreement with measurements was found assuming a model in which changes in a cell's threshold field were predicted from the calculated changes in the maximum voltage across the cell's membrane induced by the presence of a second cell. The threshold field required to electroporate a cell is changed ∼5%-10% by a nearby, nearly touching second cell. Cells aligned parallel to the porating field shield one another. Those oriented perpendicular to the field enhance the applied field's effect. In addition, we found that the dynamics of the electropermeabilization process are important in explaining observations for even our simple two-cell system.

Electroporation dependence on cell size: optical tweezers study.

Electropermeabilization or electroporation is the electrical disruption of a cell's membrane to introduce drugs, DNA/RNA, proteins, or other therapies into the cell. Despite four decades of study, the fundamental science of the process remains poorly understood and controversial. We measured the minimum applied electric field required for permeabilization of suspended spherical cells as a function of the cell radius for three cell lines. Key to this work is our use of optical tweezers to precisely position individual cells and enable well-defined, repeatable measurements on cells in suspension. Our findings call into question fundamental assumptions common to all theoretical treatments that we know of. It is generally expected that, for individual cells from a particular cell line, large cells should be easier to electroporate than small ones: the minimum electric field to cause electropermeabilization should scale inversely with the cell diameter. We found instead that each cell line has its own characteristic field that will, on average, cause permeabilization in cells of that line. Electropermeabilization is a stochastic process: two cells which appear identical may have different permeabilization thresholds. However, for all three cell lines, we found that the minimum permeabilization field for any given cell does not depend on its size.

Gene transfection of mammalian cells using membrane sandwich electroporation.

To avoid safety issues such as immune response and cytotoxicity associated with viruses and liposomes, physical methods have been widely used for either in vivo or ex vivo gene delivery. They are, however, very invasive and often provide limited efficiency. Using pEGFP and pSEAP plasmids and NIH 3T3 fibroblasts as models, we demonstrate a new electroporation-based gene delivery method, called membrane sandwich electroporation (MSE). The MSE method is able to provide better gene confinement near the cell surface to facilitate gene transport into the cells and thus shows significant improvement over transgene expression of mammalian cells compared to current electroporation techniques.