Electroporation is a widely used transfection method to introduce nuclear acids and other molecular probes into cells or tissues. Recent progress has made this method applicable to a wide range of cells and biomolecules, for in vitro transfection testing and for ex vivo and even in vivo clinical trials. The current worldwide market for electroporation is about $25 million and is expected to grow rapidly with double-digit gains over the next five years, stimulated by academia, government and pharmaceutical and biotech companies. The current commercial electroporation devices and kits (Amaxa Nucleofector technology in particular) are convenient, easy to use, and beneficial for many hard-to-transfect cell lines when non-viral gene transfer is required. However, a major drawback is that each new cell system (such as lab-specific clones or a patient's primary cells) often requires a time consuming and expensive trial-and-error search process to identify proper electroporation conditions and/or solution composition that will achieve the desired transfection level with high enough cell viability. Each cell system must be optimized individually using a finely-tuned, high electric voltage and non-uniform electric field. It would be very valuable if electroporation could be carried out in a mild, uniform electric field that is effective with various cell lines and primary cells. In this way, the need to determine new settings for each cell system can be eliminated or highly simplified. For clinical applications, there is a need to transfect a large number of cells (e.g. >109 cells) with a high transfection efficiency and cell viability. This is difficult to achieve in the current commercial electroporation device where the recommended cell population is around 105 106. We plan to design, construct and characterize a batch-type Nanonozzle Sandwich Electroporation (NSE) device and a continuous-type Converging Flow Electroporation (CFE) device. These two novel devices will then be used to investigate in vitro transfection efficiency and uptake of selected oligonucleotides and genes for cancer and stem cell applications. Their performance will be compared with the best commercial electroporation systems. Several novel electroporation devices will be developed for better in vitro and ex vivo transfection of therapeutic materials. In this study, the uptake of oligonucleotides and plasmid DNA into cancer cells and stem cells will be investigated. The proposed devices have great potential to transfect oligonucleotides and plasmid DNA into a large number of cells for clinical applications. ? ? ?
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