(161g) Corona Charge Combined with Moderate Temperature Increases for the Transfection of T-Cells In Vitro

With cellular and immunotherapies on the rise, the ability to genetically modify cells in a rapid and efficient fashion is necessary to keep up with demand. Gene electrotransfer (gene transfer by electroporation) is an appealing transfection method due to its low cost, high efficiency compared to other physical forms of gene delivery, and low toxicity compared to liposomes or viral vectors. It also allows for the genetic modification process to take place in facilities that are not equipped to handle viruses.

This study utilized an innovative way of using constant and/or pulsed corona charge and moderate temperature increases to expose cells to electric fields to initiate DNA uptake/electrotransfer into a human T-cell line (Jurkat ATCC TIB-152). This study employed the use of a novel gold dish to direct the charge to the cells without the use of electrodes, making this a non-contact and more sterile form of gene electrotransfer. Based on previous studies, there is evidence that pulsed corona charge and slightly elevated temperatures could cause a statistically significant increase in the small tracer molecule, Sytox^TM, uptake in cells. Therefore, this study investigated and optimized the delivery of the small tracer molecule Sytox^TM and then used the knowledge gained to optimize the delivery of plasmid encoding green fluorescent protein (GFP). These conditions are comprised of a treatment time, treatment temperature, and a set of corona charge parameters. These parameters are pulsed or constant corona charge application, voltage used to generate charge, and current applied to the cells. It is also hypothesized that pulsing will increase the uptake of GFP while also increasing viability of cells. Success was quantitated using viability assays, data from a fluorescent plate reader, and flow cytometric data. The result will be a novel system comprised of a gold sputter coated cell culture dish that can be used to deliver DNA to the cells and for subsequent culture and a set of treatment conditions.

The results showed that the combination of constant corona charge caused a statistically significant increase in the uptake of Sytox^TM compared to non-treated cells. Moderate temperature increases did not cause a statistically significant increase in the uptake of Sytox^TM compared to cells that were at room temperature. The combination of constant corona and moderate temperature increases caused a statistically significant increase in the uptake of Sytox^TM.

Initial experiments done with GFP were analyzed using flow cytometry. This showed that the conditions used to deliver Sytox^TM produce two morphologies of cells, a small and a large morphology. The large morphology is the bulk of the viable cell population. While the treated and/or heated cells did show an increase in the uptake of GFP, it was an increase of 0.02% of cells expressing GFP to 4%. Preliminary experimentation showed that a higher current caused a large increase in the uptake of Sytox^TM, but caused low viability in cells 48 hours post treatment. These higher currents are currently being tested to see how they impact the uptake of GFP. Using fluorescent microscope imaging, one experiment showed that if the cells survive, the majority of live cells express GFP. Work is currently being done to optimize those parameters and include comparisons to pulsed corona charge.