On the Interaction of Non-Thermal Atmospheric Pressure Plasma with Tissues S. Kalghatgi, C. Kelly, E. Cerchar, R. Sensenig, A. Brooks, A. Fridman, A. Morss-Clyne, J. Azizkhan-Clifford, G. Friedman Drexel University, 3141 Chestnut St, Bossone 404 Philadelphia, PA, USA Abstract Non-thermal atmospheric pressure plasma is now being widely developed for various clinical applications such as skin sterilization, blood coagulation, cancer treatment, angiogenesis and wound healing among others. However, understanding of mechanism of interaction between non-thermal plasma and mammalian cells is lacking. Here we investigated the possibility that the dose of non-thermal plasma can be tuned to achieve various results depending on the clinical applications ranging from enhanced cell proliferation to inducing apoptosis in malignant tissue. We also present some of the underlying mechanisms of interaction of non-thermal plasma with mammalian cells. I. INTRODUCTION Thermal plasma has been employed in medicine for coagulation and ablation for some time [1]. Treatment of tissues and cells by non-thermal plasma, where the gas temperature is nearly at room temperature, is a recent development [2]. It has been noted that non-thermal plasma applied directly to surfaces of living tissues can coagulate blood; however, it does so without charring the tissue [2, 3]. Similarly, non-thermal plasma appears to kill bacteria on the surface of living tissue without histologically visible damage [2]. It has been reported that non-thermal plasma can also mediate attachment of cells to substrates [4-6], increase transfection efficiency [7, 8] and surface sterilization [9-12]. Ability to tune non- thermal plasma effects together with the simplicity of plasma generating devices and localized nature of plasma application makes it a promising tool in medicine. However, mechanisms of interaction between non- thermal plasma and living systems have been poorly understood. Here we study the mechanisms of interaction between non-thermal plasma and mammalian cells. Several different methods of non-thermal plasma generation at atmospheric pressure are known. The type of non-thermal plasma employed in this study is called the Dielectric Barrier Discharge (DBD). It was invented by Siemens in 1859 [13]. The plasma in this discharge is created when the time-varying high voltage reaches sufficient magnitude to cause air breakdown. The presence of dielectric layer (dielectric barrier) in the path of the discharge limits its current which, in turn, limits the energy transferred to ions and neutral gas species keeping their temperature low. Although the plasma gas temperature is low, the presence of charged particles, radicals and electronically excited molecules and atoms makes DBD plasma a potentially active medium whose properties can be controlled to some extent through gas composition as well as waveform of the time-varying applied voltage. II. METHODS AND MATERIALS Mammalian Breast Epithelial Cells (MCF10A) were maintained in high glucose Dulbecco’s Modified Eagle’s Medium-Ham’s F12 50:50 mixture (DMEM-Ham’s F12 50:50) (Cellgro, Mediatech, VA, USA) supplemented with 5% donor horse serum (Sigma Aldrich, St. Louis, MO, USA), Epidermal Growth Factor (EGF, 100 µg/ml, Sigma Aldrich, St. Louis, MO, USA), Hydrocortisone (1 mg/ml, Sigma Aldrich, St. Louis, MO, USA ), Cholera Toxin (1 mg/ml), Insulin (10 mg/ml, and Penicillin/Streptomycin (500 μl, 10000 U/ml penicillin and 10 mg/ml streptomycin). Media was changed every two days. For plasma treatment, cells were washed with phosphate buffered saline (PBS), detached with 0.25% trypsin (GIBCO, Invitrogen, CA, USA), and seeded near confluence (4 x 10 5 cells/well) on 22 x 22 mm square glass cover slips (VWR, PA, USA ) in 6-well plates (Greiner Bio One, NC, USA). Cells were cultured for 24 hours prior to plasma treatment in 2.0 ml supplemented media in a 37°C, 5% CO 2 incubator to allow full attachment and spreading. N-Acetyl-L-cysteine (2 mM, Sigma-Aldrich, St Louis, MO), an intracellular reactive oxygen species (ROS) scavenger and sodium pyruvate (10 mM, Sigma-Aldrich, St Louis, MO), an extracellular ROS scavenger were used to block the reactive oxygen species produced by non- thermal plasma treatment. A. Plasma Treatment Non-thermal atmospheric pressure dielectric barrier discharge plasma was produced using an experimental setup similar to one previously described and schematically illustrated in Figure 1 B. The non-thermal plasma was generated by applying alternating polarity 1133 9781-4244-4065-8/09/$25.00 ©2009 IEEE