Micronozzle Array Enhanced Sandwich Electroporation of Embryonic Stem Cells

Abstract

Electroporation is one of the most popular nonviral gene transfer methods for embryonic stem cell transfection. Bulk electroporation techniques, however, require a high electrical field and provide a nonuniform electrical field distribution among randomly distributed cells, leading to limited transfection efficiency and cell viability, especially for a low number of cells. We present here a membrane sandwich electroporation system using a well-defined micronozzle array. This device is capable of transfecting hundred to millions of cells with good performance. The ability to treat a small number of cells (i.e., a hundred) offers great potential to work with hard-to-harvest patient cells for pharmaceutical kinetic studies. Numerical simulation of the initial transmembrane potential distribution and propidium iodide (PI) dye diffusion experiments demonstrated the advantage of highly focused and localized electric field strength provided by the micronozzle array over conventional bulk electroporation.

Microfluidics cell electroporation

“…In a series of articles, Fei et al (2007Fei et al ( , 2010 proposed the membrane sandwich technique (MSE) for cell electroporation. This method suggested immobilizing and sandwich the cells between two polyethyleneterefthalate (PET) membranes to improve the cell transfection and viability.…”

Micro-/nanofluidics based cell electroporation

“…To firmly hold cells, a gentle vacuum was sometimes used to pull a small portion of cells inside the microscale constrictions. [46][47][48][49][55][56][57][58] If cell lysis is necessary, more efficient lysis electrode designs such as interdigitated electrodes with a saw-tooth structure could be used. Besides trapping cells with physical contact, optical tweezers technology (a technology that creates radiation pressure with an intensified laser beam to facilitate non-contact optical trapping to colloids) was also introduced to help grab and relocate cells remotely in electroporation micro-devices ( Fig.…”

“…46,55,56,63,75 By sandwiching cells ($10 4 ) between two pieces of track-etched membrane consisting of thousands of micropores, we successfully transfected 3T3 cells and mouse embryonic stem (mES) cells with better transfection efficiency ($2-3 folds increase) when imposing low-voltage pulses (<1-35 V). 57,58 An alternative array-type system used a 96-well format to electroporate liquid droplets (10-20 ll) encapsulated with molecule probes and cells. 65 With a multiple channel robotics, they effectively delivered dextran, siRNA, and cDNA into primary neurons, differentiated neutrophils, and some hard-to-transfect cells (e.g., HL-60) at a speed of $10 6 cells/min.…”

Recent Trends on Micro/Nanofluidic Single Cell Electroporation

“…The permeabilization area can be controlled with the pulse amplitude and the degree of permeabilization can be controlled with the duration of pulses, numbers of pulses, where longer pulses provide a larger perturbation area in the cell membrane [34,35]. In earlier studies of micro/nanofluidic based single cell electroporation, authors analyze cellular content and cellular properties [36][37][38][39], transfection of cells [17,[40][41][42] and inactivating cells [43][44][45] with the use of micro-channel based electroporation [46][47][48][49], micro-capillary based electroporation [50][51][52], electroporation with solid microelectrode [36,[53][54][55], membrane sandwich based microfluidic electroporation [56,57], microarray single cell electroporation [58], optofluidic based microfluidic devices [59][60][61][62][63][64][65], etc. Table 1 describes in detail micro/nanofluidic based single cell transfection, cell lysis, cell type with species, potential difference, pulse duration, etc.…”