(333a) Modification of Cells Using a High-Throughput Microelectroporator

Genetic modification of cells to redirect their specificity towards desired antigens expressed on target cells is a promising therapy for the treatment of diseases including cancer. However, conventional viral and especially non-viral gene transfer methods for gene transfer are laborious, inefficient, and rely on full-scale good manufacturing practice GMP (GMP) facilities. There is a current trend to produce micro and nano-size devices that can perform physical, chemical, and biological processes on a small scale with the same efficiency as conventional macroscopic systems. We have designed a microfluidic/electrotransfer system for continuous insertion of transgenes coded by RNA and/or DNA into cells via electroporation. Electroporation is an indispensable and safe method for delivery of drugs and genes into cells and has been used in clinical trials1,2. Our device is capable of handling a large number of cells, on the order of those needed clinically for therapy. Further, it can operate in a large parameter space, optimizing frequency, amplitude, waveform shape, and number of pulses experienced. By optimizing the electroporation parameters, external mRNA or DNA will be incorporated into a cell, enabling the cell to produce its own drugs. Finally, the components of the device are adaptable to be implemented in a clinical setting. Poration occurs when the transmembrane potential across a cellular membrane is greater than a threshold potential. The membrane potential is approximately given by Ed, where E is the electric field and d is the membrane thickness3. Once the membrane has porated, mRNA or DNA passes into the cell and the desired function is expressed. Our device was fabricated using standard photolithographic techniques, shown in Figure 1.Jurkat cells at a concentration of about 106 cells/mL were flowed through our device at a rate of 2 μL/s. Propidium iodide (PI), a nucleic acid stain, at a 1:10 dye/media volume ratio was used to stain cells to determine viability and poration after exposure. In Figure 2, we show how flow through the device and exposure to PI affects cell viability. Compared to the control population, we see no significant cell death (less than 10%) on the time scale required for electroporation. This demonstrates that tens of minutes of exposure to PI or to high sheer stress does not cause cellular death, and that a clinically relevant number of cells pass successfully through the device. For experiments showing electroporation, we used an optimization procedure described by Canatella and Prausnitz in order to determine the electric pulse amplitude, duration, and number of pulses each cell should be is exposed to for optimal electroporation4. We selected 10 millisecond square wave pulses at 100 Hz and a field of 833 V/cm to porate Jurkat cells. The flow velocity sets the number of pulses each cell sees to 12. Figure 3 shows a collection of cells that have passed through the electrode and are fluorescing, indicating that PI has entered the cell. Our next experiments are to check cell viability after electroporation. We have shown a clinically relevant number of cells may pass through our electroporator without cell death and are modified successfully. REFERENCES: 1Costa et al. A method for genetic modification of human embryonic stem cells using electroporation. Nature protocols (2007) vol. 2 (4) pp. 792-6 2Van Tendeloo et al. Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells. Blood (2001) vol. 98 (1) pp. 49-56 3Krassowska et al. Modeling Electroporation in a Single Cell. Biophysical Journal (2007) vol. 92 (2) pp. 404-417 4Canatella et al. Prediction and optimization of gene transfection and drug delivery by electroporation. Gene therapy (2001) vol. 8 (19) pp. 1464-9