Ie Ingrid Kieft

Plasma needle for in vivo medical treatment: recent developments and perspectives

In this paper we describe the hitherto unravelled facts on the interactions of a cold atmospheric plasma with living cells and tissues. A specially designed source, plasma needle, is a low-power discharge, which operates under the threshold of tissue damage. When applied properly, the needle does not cause fatal cell injury which would result in cell death (necrosis). Instead, it allows precise and localized cell removal by means of the so-called cell detachment. In addition, plasma can be used for bacterial disinfection. Because of mild treatment conditions, plasma disinfection can be performed in vivo, e.g. on wounds and dental cavities. Presently, one strives to obtain a better control of the operating device. Therefore, plasma has been characterized using a variety of diagnostics, and a smart system has been designed for the positioning of the device with respect to the treated surface.

Superficial treatment of mammalian cells using plasma needle

Interactions of a small-size, non-thermal plasma (plasma needle) with living cells in culture are studied. We have demonstrated the non-destructive character of the plasma needle: under moderate conditions (low-power and low concentration of molecular species) the plasma needle does not heat biological samples and does not induce cell death. Treatment of living cells is restricted to the cell exterior (membrane). As a result of the interactions of plasma radicals with cell adhesion molecules, cell attachment is temporarily interrupted; the loose cells can be removed, reattached or transferred. This effect may prove very useful in fine surgery, where a part of the tissue must be removed with high-precision, without damage to the adjacent cells and without inflammatory reaction.

Reattachment and Apoptosis After Plasma-Needle Treatment of Cultured Cells

Nonthermal plasmas can be used to locally influence cell adhesion: cells can be removed from their surroundings without causing necrosis. In fact, cells remain alive and can reattach within hours. This phenomenon may, in the future, be used for microsurgical procedures. Another method to remove cells is to induce apoptosis or programmed cell death. This type of cell death is preferred above necrosis, which may cause inflammation reactions. When the detached cells are allowed to reattach and grow, it is important to know their condition. Therefore, long-term effects of plasma-needle treatment were assessed, with special focus on reattachment and apoptosis. The cells were treated using a plasma needle. This device generates a small (1-mm diameter) plasma at atmospheric pressure. To avoid any heat effects, it is important that the plasma temperature is at or below physiological temperature. This is the case for the plasma needle

Plasma treatment of mammalian vascular cells: a quantitative description

For the first time, quantitative data was obtained on plasma treatment of living mammalian cells. The nonthermal atmospheric discharge produced by the plasma needle was used for treatment of mammalian endothelial and smooth muscle cells. The influence of several experimental parameters on cell detachment and necrosis was tested using cell viability assays. Interruption of cell adhesion (detachment) was the most important cell reaction to plasma treatment. Treatment times of 10 s were enough to detach cells in the cultured cell sheet. Under extreme conditions, cell necrosis occurred. Cell detachment without necrosis could be achieved at low voltages. It was shown that the thickness of the liquid layer covering the cells was the most important factor, which had more influence than treatment time or applied voltage. The results show no remarkable differences between the responses of the two cell types.

The effects of UV irradiation and gas plasma treatment on living mammalian cells and bacteria: a comparative approach

Living mammalian cells and bacteria were exposed to irradiation from narrow-band UV lamps and treated with a nonthermal gas plasma (plasma needle). The model systems were: Chinese Hamster Ovary (CHO-K1) cells (fibroblasts) and Escherichia Coli bacteria. UV irradiation can lead to cell death (necrosis) in fibroblasts, but the doses that cause such damage are much higher than those needed to destroy Escherichia Coli. The usage of UV radiation in combination with active oxygen radicals lowers the UV dose sufficient to kill the cells. However, in any case the fibroblasts seem to be fairly resistant to UV radiation and/or radicals. Possibly, the lamps may be used for decontamination of infected wounds. The most important active species in an atmospheric plasma are the radicals; the role of UV is less pronounced. Treatment of CHO-K1 cells with the plasma needle can lead to cell necrosis under extreme conditions, but moderate doses cause only a temporary interruption of cell adhesion. Plasma needle may be used for fine tissue treatment (e.g., controlled cell removal without inflammation) and also for bacterial decontamination.

Plasma interactions with living cells

Summary form only given. In pursuit of minimum-invasive surgery one has to develop techniques, that allow specific cell removal or rearrangement without influencing the whole tissue. In conventional or laser surgery individual cells undergo accidental cell death (necrosis), which is followed by inflammation and may lead to permanent tissue damage. In contrast, cold plasma techniques allow cell removal without necrosis. At the Eindhoven University a suitable small-size plasma source has been developed (plasma needle) and several potentially beneficial plasma-cell interactions have been identified. These reactions include: cell detachment without affecting cell viability, induction of apoptosis (programmed cell death), and altering cell proliferation rate. So far the tests have been performed on cells in culture (mouse fibroblasts or human epithelial cells of lung carcinoma), but recently we have introduced a new model: tissue engineered skin. The effect of plasma-induced cell detachment has been already identified in this model. At present we check for early markers of tissue damage and differentiation of keratinocytes after plasma treatment. In parallel, we continue the study on mouse fibroblasts. MTT assay for long-term viability has been performed. Cell proliferation rate has been monitored using the BrdU assay (a marker for newly formed DNA). A strong link between the plasma properties and triggered cell reactions is expected. Moderate cell damage, which leads to (reversible) detachment or apoptosis, may result from interactions with plasma-produced radicals. The ROS (reactive oxygen species) are known to play an important role in these processes. We have shown that plasma radicals can exist in the liquid phase (cell culture medium). Micro-molar concentrations of ROS from the plasma have been detected using a fluorescent probe in combination with (confocal) LIF, and correlated with gas-phase plasma properties. We conclude that ROS concentrations are within the safe range: the radicals can trigger specific cell reactions, but are unable to kill the cells.

Power outflux from the plasma: an important parameter in surface processing

In this work we characterize a low-power radio-frequency atmospheric plasma (plasma needle) in terms of dissipated (input) and emitted power per unit surface (power outflux). The plasma is a non-thermal source, used for treatment of biological tissues and other vulnerable surfaces. A calibrated thermal probe is used to determine the power emitted from the plasma towards treated surfaces. Transmission of the emitted plasma power through various media (solid layers, fluids and physiological media) is studied for a broad range of plasma conditions. These data give insight into various contributions to the power outflux (thermal conduction, radiation and energetic species), as well as the penetration depth of the plasma into treated objects. The power outflux is shown to be a very important parameter, which determines the performance of the plasma tool. For the effectiveness and reproducibility of the process the power outflux is much more important than the nominal power setting. Thus, a thermal probe should become a standard control unit in surface processing reactors.

UV Excimer Lamp Irradiation of Fibroblasts: The Influence on Antioxidant Homeostasis

UV excimer lamps are efficient and economical narrowband sources of UVB and UVC radiation. Potentially, these lamps may be used in bacterial disinfection or in minor cosmetic procedures, but safety tests are necessary. For this purpose, cellular damage induced by the UV radiation is assessed. 3T3 mouse fibroblasts are used as a model system. It is found that lethal damage occurs above a certain wavelength-dependent threshold dose. Furthermore, the antioxidant balance of the cells is influenced; glutathione levels are usually depleted, both under and above the lethal threshold. A striking increase of glutathione level is observed after UVC irradiation. This is attributed to an enhanced repair activity as a response to acute oxidative stress induced by UVC. In contrast to UVC, low-power irradiation with UVB seems to induce very little cellular damage

Towards Plasma Surgery: Plasma Treatment of Living Cells

The physical, biological and technical background for high-precision plasma surgery is prepared in a multi-disciplinary team. The aim of the research is to achieve controlled removal of diseased cells and bacteria without harming the healthy rest of the tissue. For this purpose, a small, cold, flexible and non-toxic plasma is developed (the plasma needle) and tested on cultured cells and bacterial samples. The needle is an atmospheric discharge induced by a radio-frequency voltage applied to a metal pin. This plasma operates at room temperature, in the milliwatt power regime; it poses no risk of thermal or electrical damage to living tissues. Several beneficial responses of living cells to plasma treatment have been already identified. Plasma does not cause accidental cell death (necrosis), which leads to inflammation and tissue damage. Instead, it allows to detach cells from each other and from the scaffold, and thus to remove them in a non-destructive way. Moreover, plasma is capable of bacterial inactivation. I parallel, we have determined the electrical and optical properties of the plasma and found a method of precise positioning of the plasma needle with respect to the treated tissue.