SAMEER KALGHATGI

           

This page gives an overview about my research on understanding the mechanisms of interaction of non-thermal atmopsheric pressure dielectric barrier discharge plasma with mammalian cells.

Non-Thermal Plasma Interaction With Cells

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

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.

Methods MCF10A cells on glass cover slips were exposed to non-thermal plasma at various doses from 0.13 J/cm2 to 7.8 J/cm2 (Figure 1). Briefly, each cover slip was removed from the 6-well plate and placed on a microscope slide, which was then positioned on the grounded base of the plasma device. 100 µl of supplemented media was added to the glass cover slip before plasma treatment to prevent sample drying. Following plasma treatment, the cells were held in the treated medium for one minute and then the cover slip was placed in a new 6-well plate, 2 ml of supplemented media was added to the well, and the samples were returned to the incubator for one hour before analyzing the samples using immunofluorescence or western blot.

Figure 1. Schematic of setup for non-thermal plasma treatment of mammalian cells in vitro

 

Three different approaches were used for non-thermal plasma-treatment of cells in vitro: direct, indirect and separated. In direct treatment, the sample itself was one of the electrodes that created the plasma discharge, as illustrated in. Plasma discharge occurred between the powered high voltage electrode quartz surface and the sample surface, which exposed the sample directly to both neutral reactive species and charged particles. In contrast, for indirect treatment, a grounded mesh was placed between the high voltage electrode and the treated sample to prevent charged particles from reaching the sample surface. In separated plasma treatment, medium alone was plasma treated separately from cells and then immediately applied to cells. In this case, cells were not in direct contact with any plasma component.

Results

 

 

 

 

 

 

 

 

 

 

 

 

 

 


 

 

Figure 6. Cells were subjected to DBD plasma as described earlier (direct, D) or media (100 ml) was subjected to DBD plasma and then transferred to the cells (separate, S).

Figure 4. Cells were treated with the indicated dose of DBD plasma; one day after treatment, 300 cells were plated in a 6 cm dish and colonies were counted after 8 days. Data from triplicate samples (±S.E.M.) are expressed relative to the # of colonies in the untreated control.

Figure 3. Cells were treated with the indicated dose of DBD plasma as described.  3 days after treatment, cells were harvested and stained with Annexin V/ propidium iodide (PI) and analyzed by Guava. 

Figure 2. Fold growth is enhanced in non-thermal plasma treated cells 5 days after treatment. Plasma treated cells were counted using a Coulter counter 1 and 5 days after treatment. * p < 0.01 as compared to control.

Figure 5. Cells were subjected to DBD plasma as described earlier (direct, D) or media (100 ml) was subjected to DBD plasma and then transferred to the cells (separate, S).

Figure 8. Cells were subjected to DBD plasma as described earlier (direct, D) or media (100 ml) was subjected to DBD plasma and then transferred to the cells (separate, S).

Figure 7. Cells were subjected to DBD as described earlier (direct, -) or a grounded mesh was placed between the electrode and the medium (indirect, +).

Figure 9. MCF10A cells were incubated for 1 hour with 4 mM N-acetyl cysteine (NAC) (+) or cell culture medium (-), followed by treatment with the indicated dose of DBD plasma. g-H2AX (upper panel) or a-tubulin (lower panel) was detected by immunoblot of cell lysates prepared one hour after plasma treatment.

 

 

 

Conclusions: Use of DBD plasma for clinical applications requires an understanding of its interaction with living tissues.  We have shown here that non-thermal plasma interacts with cells indirectly by modifying the surrounding environment of the cells during treatment. We also confirmed that long living reactive oxygen species (ROS) produced by plasma in cell culture medium mediate interaction between non-thermal plasma and mammalian cells. The amount of ROS produced by plasma can be tightly controlled by varying the applied voltage, allowing fine tuning of therapeutic effect, from stimulating cell proliferation to inducing apoptosis.  Future work will focus on establishing whether the effects of plasma are through membrane lipid peroxidation or due to uptake of long living organic hydroperoxides by active transport mechanisms in the cells. The potential clinical applications of DBD plasma include treatment of wounds to enhance healing and sterilize wound surfaces or controlled ablation of tissue, including benign lesions or cancers.

References

  1. Vargo, J. J. Gastrointest Endosc 59, 81 (2004).

  2. Fridman, G., et al., Plasma Chemistry and Plasma Processing 26, 425 (2006). Kalghatgi, S., et al., Plasma Science, IEEE Transactions on 35, 1559 (2007).

  3. Kalghatgi, S., et al., Plasma Science, IEEE Transactions on 35, 1559 (2007).

  4. Detomasoa, R., D'Agostinoa, R., Senesi, G., and Favia, P., Surface and Coatings Technology 2000, 1022 (2005).

  5. Gristina, E., Senesi, G., Milella, A.,  Nardulli, M., Sardella, E., Favia, P.,  D'Agostino, R., Journal of Biomedical Materials Research Part B: Applied Biomaterials 88B, 139 (2008).

  6. Rosso, F., et al., J Cell Physiol 207, 636 (Jun, 2006).

  7. Coulombe, S., 1st International Conference on Plasma Medicine (ICPM), Corpus Christi, TX, 2007.

  8. Coulombe, S., Léveillé, V., Yonson, S. and Leask, R. L., Pure and Applied Chemistry 78, 1147 (2006).

  9. Laroussi, M., Mendis, D., and Rosenberg, M., New Journal of Physics 5, (2003).

  10. Laroussi, M., Alexeff, I., Kang, W., Plasma Science, IEEE Transactions on 28, 184 (2000).

  11. Laroussi, M., Richardson, I., Dyer, J., IEEE Transactions on Plasma Science 30, 158 (2002).

  12. J. R. Roth, S. Nourgostar, and T.A. Bonds, IEEE Transactions on Plasma Science 35, 233 (2007).

  13. Siemens, C. W., Journal of the Franklin Institute 74, 166 (1862).

Relevant Publications

  1. Effects of Non-Thermal Plasma on Mammalian Cells, S. Kalghatgi, C. Kelly, E. Cerchar, B. Torabi, O. Alekseev, A. Fridman, G. Friedman, J. Azizkhan-Clifford, PLoS ONE, 2010. 6(1): e16270. doi:10.1371/journal.pone.0016270. FULL TEXT (PDF)

  2. Low Dose Non-Thermal Plasma Interacts with Mammalian Cells Indirectly through Modification of the Cell Culture Medium, Sameer Kalghatgi, C. Kelly, E. Cerchar, A. Fridman, J. Azizkhan-Clifford, G. Friedman, To appear In Abstracts and full papers 19th International Symposium on Plasma Chemistry, Jul 26th - July 31st 2009, Bochum, Germany

  3. 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. 17th IEEE Pulsed Power Conference (PPC), Jun 29th - Jul 2nd 2009, Washington D.C., USA

  4. Comparison of Different Non-Thermal Plasma Treatments of Mammalian Cells, S. Kalghatgi, C Kelly, E. Cerchar, A Fridman, J Azizkhan-Clifford, G Friedman, 36th IEEE International Conference on Plasma Science (ICOPS), May 29th - Jun 4th 2009, San Diego, California, USA.

  5. Interaction of Non-Thermal Dielectric Barrier Discharge Plasma with DNA inside Cells (Contributed talk and selected as finalist for Student Paper Excellence Award), S. Kalghatgi, C. Kelly, G. Fridman, J. Azizkhan, A. Fridman and G.Friedman. 61st Annual Gaseous Electronics Conference, Oct 13th-Oct 17th, Dallas, Texas, USA

  6. Penetration of Direct Non-Thermal Plasma Treatment into Living Cells (Contributed Talk), S. Kalghatgi, C Kelly, G Fridman, A Fridman, J Azizkhan-Clifford, G Friedman, 35th IEEE International Conference on Plasma Science (ICOPS), June 15-19 2008, Karlsruhe, Germany.

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