Gene Delivery to Embryonic Stem Cells by Micronozzle Array Enhanced Sandwich Electroporation

Electroporation is one of the most popular non-viral gene transfer methods for embryonic stem cells. Commercial bulk electroporation techniques, however, require high electrical field and provide non-uniform electrical field distribution among randomly distributed cells, leading to limited transfection efficiency and cell viability, especially for low number of cells. We present here a membrane sandwich electroporation (MSE) system using well-defined micronozzle array. This device is capable of transfecting hundreds to millions cells. By adjusting the laser output powers and laser beam focus points, we are able to produce both converging micronozzle and straight microchannel arrays on the membrane. This new design was used for gene transfection of mouse embryonic stem cells (mESCs). Because of the highly focused and localized electric field strength, we observed higher and more uniform gene transfection with better cell viability of mESCs comparing to the bulk electroporation. The ability to treat a small number of cells (i.e. a hundreds) offers great potential to work with hard-to-harvest patient cells for pharmacokinetic studies. Experimental observation of cells and gene under electroporation using spin-disk dynamic confocal microscopy and numerical calculation of transmembrane potential explain the observed differences between MSE and bulk electroporation.