Barbara A. Oakley, Darrin M. Hanna, Sachin Kandlikar, Brooks Gross, Gabrielle A. Stryker

1. Power and Energy Required to Produce Cell Mortality in SWLA-2 Hybridomas with 1 kHz Millisecond Range Pulses Barbara A. Oakley, Darrin M. Hanna,Sachin Kandlikar, Brooks Gross,Gabrielle A. Stryker

2. Abstract This paper describes the initial experimental setup and results involving the percentage cell lysis in SWLA-2 murine hybridomas produced by AC electric field pulses at 1kHz with pulse widths ranging from 1 ms to 1 second. Cells were cultured and separate samples examined at 48 hours to determine cell mortality.

3. The effects of electric fields on biological cells has proven useful for a wide range of applications in recent decades • In vivo delivery of molecules and genetic materials to eukaryotic cells has been carried out using electropermeabilization techniques with field strengths of less than lethal magnitudes. • These techniques have also been used for cell-cell fusion, such as the production of monoclonal antibodies. • As the fields can effectively kill bacteria and yeasts, pulsed electric fields of lethal magnitudes have proven useful for food preservation and the reduction of biofouling. • Relevant to these research efforts is the ongoing investigation of the effect of the rapidly pulsed fields produced by Tasers on the human body.

4. Motivation The studies described in this paper, using AC pulses of varying pulse width and frequency, were performed to gain an understanding of the parameters for the lysing of eukaryotic cells using a pulse of AC electric field.

5. Cell Line • The cell line used in this experiment was the SWLA-2 murine hybridoma (HB12560), purchased from ATCC. The cells were grown at 37 °C and 5% CO2 in a water-jacketed CO2 incubator. Hybridomas were grown in serum free media, (Gibco). 2) Preparation: The hybridomas used in these experiments were prepared as follows prior to the experiments. The total number of cells was obtained by counting them in Neubauer's hemocytometer using Trypan blue stain 0.2%. Cells were spun down at 130 × g for 10 min. The supernatant was discarded and the pellet was re-suspended at 3 x 105 cells/ml.

6. Electrical Equipment and Cuvettes • Sinusoidal electric field pulses of varying pulse length (1 ms to 1 second), amplitude and frequency were delivered to the cell suspensions by a lab-built setup was capable of providing approximately 500 W with an rms voltage maximum of approximately 90 V. Cuvettes were standard 1 mm gap width, 100 ml electroporation cuvettes.

7. Cell Counting • For all of the experiments conducted, the cells inoculated into 3 replicate wells of a 96-well plate (30 µl cells/well) containing 170 µl of culture media. Each experiment was then repeated on a subsequent sample (cuvette) and added into 3 replicate wells of second 96 well plate. The number of viable cells was determined by counting the number of live cells in Neubauer’s hemocytometer with trypan blue staining. The cells were counted 48 hours after exposure to the fields. The cell counts were relative to control cells that had not been exposed to electric fields.

8. Results

9. Discussion • Using the SWLA-2 (hybridomas) approximate diameter of 15 microns, a root mean square membrane potential of ΔVmrms = 1.0 V would require that the voltage across the cuvette was 44.44 Vrms. Clearly this voltage across the cuvette was exceeded, at least for AC pulses of 100 ms or less. However, the AC pulse at higher voltages apparently did not last long enough to produce extensive cell lysis until extended pulse lengths ( 100 ms) were used. In normal electroporation experiments, lysis is induced when the fields are held across the cell membranes long enough for the membrane lipids to rearrange, leading to pores which cause cell death. In the experiments discussed here, however, heating may have contributed to cell lysis.