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2004 - Gas Plasma Effects On Living Cells - Stoffels
2004 - Gas Plasma Effects On Living Cells - Stoffels
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Fig. 1. A typical appearance of the plasma glow: left–in the unipolar mode (plasma is confined at the tip of the needle) and right–in the bipolar mode
(plasma stretches towards the grounded object, in this case skin).
containing dish and treated with the plasma under the released. The latter causes an inflammatory reaction and
mildest conditions (voltage of 190 V, distance needle to damage to the tissue, so it must be avoided in refined
droplet of 1 mm). Afterwards, the dishes have been treatment.
incubated and 24 to 48 h later the newly formed colonies In our experiments we have used various types of cells:
have been counted. Counting the colonies allows to verify epithelial-like fibroblasts of Chinese hamster ovary (CHO-
the ability to proliferate and survive on long term, so it K1), mouse fibroblasts (3T3) and human epithelial cells of
provides more useful information than observing indivi- lung carcinoma (MR65). These cells are good model
dual bacteria. Therefore, colony counting is the most systems from the point of view of future applications:
popular method of determining the bacterial activity. The skin treatment and wound healing.
exact protocols can be found elsewhere [9]. In Fig. 4 a Cells in culture usually form a two-dimensional sheet, as
typical survival curve is shown. The D-values deduced for shown in Fig. 5. They make contacts with their surround-
the plasma needle are of course higher than those for large, ings by means of trans-membrane proteins, called cell
high-power reactors: the time needed to inactivate 90% of adhesion molecules (CAMs): cadherins and integrins.
E. Coli is about 40 s. On the other hand, the low-power Cadherins connect the cells with each other, while integrins
needle is much more suitable for treatment of small dental attach the cells to the bottom.
cavities, without heating and damage. Moreover, it causes During plasma treatment, cells are subjected to more
depth sterilisation of bacteria hidden in their normal severe conditions than bacteria—voltages of at least 300 V
medium. The actual bacteria causing dental decay are are needed to induce any reaction. The exposure time is 30–
Streptococcus Mutans; they are much more vulnerable than 60 s. The cells are covered by physiological saline solution,
E. Coli, and therefore very difficult to be kept alive outside to prevent them from desiccation and to divert static
the oral cavity. The experiments with E. Coli give electricity (the latter may cause membrane charging, as in
confidence that the needle will allow to inactivate the case of dry bacterial samples). The thickness of the liquid
Streptococcus. For practical purposes, 99% of the bacteria film is 0.3–0.5 mm, the distance from the needle to the
in a dental cavity must be eliminated [10]; this corresponds liquid surface is 1 mm. After treatment, cells are observed
to a treatment time of 1–1.5 min. Afterwards, the cavity under a conventional phase-contrast or a confocal fluor-
opening should be sealed with a very small filling. As said escence microscope. Cell viability is assayed using fluor-
before, the treatment is painless. escent staining: propidium iodide (PI) to detect dead
In the next section it will be shown that mammalian cells (necrotic) cells and cell tracker green (CTG) to detect
are much more resistant to plasma treatment than bacteria. living cells. The exact procedure is described elsewhere [11].
Thus, plasma treatment is a very promising method for in Generally, no necrosis is observed: cells remain alive after
vivo sterilisation, where bacteria are eliminated while the plasma treatment. However, the interactions between them
body cells remain unharmed. are interrupted. This is a general feature, observed in all
studied cell types. A cell, which has lost the contact
through CAMs, changes its shape from elongated to
4. Plasma treatment of cells in culture spherical (see Fig. 6). Such detachment is initiated by the
plasma and it commences for about 20 s; under the
Plasma interactions with eukaryotic cells are much more
microscope it can be seen that the layer of cells ‘‘dissolves’’.
complicated than interactions with bacteria. The cells are
Cells remain in this state for several hours (2–6 h);
generally more resistant, and can have many interesting
afterwards the cell contact is restored. We expect that
responses of self-defence. Some of these responses are of
interest for refined, high-precision treatment of diseased plasma species affect only the CAMs, while the cells remain
healthy and capable of replacing the damaged proteins.
tissues. Therefore, we conduct a fundamental study in
This kind of plasma action can be seen as ‘‘surface
order to identify and elucidate all possible reactions of
living mammalian cells to plasma treatment. Desired
effects are controlled cell removal or manipulation without
membrane damage. Membrane rupture results in so-called
accidental cell death (necrosis), where the cytoplasm is
Fig. 6. Left: a CHO-K1 after plasma treatment. The detached cells are round. Right: a 3T3 cell sample just seconds after treatment. The sheet dissolves
and the cells drift away, leaving an empty space on the substrate.
processing’’ of cells. The precision of treatment is very applicable effects have been identified, but the research is
high: the area of detached cells can be as small as 0.1 mm in still in the introductory/testing phase. Most of the studies
diameter. The borders between the ‘‘detached’’ and are of fundamental nature and they involve rather simple
uninfluenced areas are as sharp as a single cell thickness model systems. However, the first results show that plasma
ð20 mmÞ: Potentially, cell detachment is the finest way of cell treatment has the potential of becoming a valuable method
manipulation and it may become a minimum-destructive in the surgery of the future. Bacterial decontamination
method of disposing of unwanted cells. without harming the tissue, control of inflammation,
The detailed mechanisms of bacterial decontamination treatment of skin, wounds and caries are just a few
or cell detachment due to interactions with the plasma examples of possible applications. We are positive that
needle are not yet completely clear. In both cases bacteria/ more sophisticated therapies are about to emerge.
cells are always suspended in/covered by physiological
solutions. Nevertheless, we observe that plasma influence
reaches quite deep under water: the effects on cells are
References
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