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Physica Scripta. Vol. T107, 79–82, 2004

Gas Plasma Effects on Living Cells

E. Stoffels
, R. E. J. Sladek and I. E. Kieft
Eindhoven University of Technology, Department of Biomedical Engineering, P.O. Box 513, 5600 MB Eindhoven, The Netherlands

Received September 18, 2003

PACS Ref: 52.80.Pi, 87.16.Dg

Abstract high to treat tissues, the development of a suitable plasma

This paper surveys the research activities at the Eindhoven University of
tool is the first experimental challenge in this multi-
Technology (The Netherlands) in the area of biomedical applications of gas disciplinary research.
discharge plasmas. A non-thermal atmospheric plasma source (the plasma In this paper the biomedical plasma research at the
needle) has been developed, and its interactions with living mammalian cells Eindhoven University of Technology will be surveyed.
and bacteria are studied. It is concluded that plasma can efficiently kill bacteria
First, the novel non-thermal atmospheric plasma source
without harming the cells, and also influence the cells without causing cell
death (necrosis). In future it will lead to applications like skin (wound) and
(‘‘plasma needle’’) will be described. Special attention will
caries treatment. be given to bacterial inactivation, because it is intended
that the plasma needle become a novel tool for painless and
tissue-saving treatment of dental caries. Furthermore, fine
1. Introduction plasma interactions with living cells will be described, and
Surface processing of solid state is one of the most the possibility of removing cells without harming the tissue
important applications of gas discharges (plasmas). Chem- will be discussed.
ically active but non-destructive, versatile but selective,
plasmas are capable of virtually any surface treatment:
etching, deposition, cleaning and surface activation. Since 2. The plasma needle
many plasmas are non-thermal (operating at room or
In this section the novel small-size plasma source for fine
slightly elevated temperature), they are perfectly suited for
biomedical treatment will be briefly introduced; a detailed
fine treatment of heat-sensitive (organic) materials.
description can be found elsewhere [4]. The discharge type is
Recently, plasma techniques have been introduced in
an atmospheric radio-frequency (RF) glow. In case of RF
biomedical sciences. Plasma coating of artificial implants
excitation, the active plasma zone is created only at the
to increase their bio-compatibility [1], surface micro-
powered electrode; a bipolar configuration is not necessary.
patterning of certain substrates to control cell adhesion
In our arrangement the powered electrode is a thin (0.3 mm)
[2], bacterial decontamination of medical/surgical equip-
tungsten wire, the remote surrounding acts as the counter-
ment [3] are just a few examples illustrating the rapidly
electrode (ground). In the vicinity of a treated object (tissue),
rising popularity of plasma technology in medicine. The
its surface acts as a grounded electrode and the plasma glow
next step is applying plasmas in vivo to achieve a desired
spreads over it. These two situations: a unipolar glow and a
modification of the living tissue. Of course, plasmas will
bipolar needle-to-tissue discharge are illustrated in Fig. 1.
not be used for bulk surgery. Contrariwise, their major plus
The scheme of the setup is shown in Fig. 2.
point is the finesse: superficial action and minimum damage
In this type of discharges, voltage fall occurs in the
to the tissue. The applications are numerous. Sterilising
closest vicinity of the powered electrode; in the outer
properties of discharge plasmas may eliminate/reduce
plasma zones, which make contact with biological samples,
infection and aid wound healing, or enable treatment of
only small rest electric fields are present. This guarantees
dental caries without drilling. Plasmas are also capable of
no electric hazard during treatment. Dependent on the
fine modification of living cells, e.g., inducing programmed
desired chemical effect, various gases can be used: air,
cell death, ‘‘dissolving’’ the tissue without damaging the
nitrogen, hydrogen and many others. However, it is
cells, and reducing the proliferation activity. These effects
recommended that the buffer gas be Helium. There are
may prove extremely useful when high-precision cell
many advantages of using Helium: the operating voltages
removal or preventing uncontrolled tissue growth is
are lower than in other gases, the gas temperature is low
required.
due to high thermal conductivity of Helium. Moreover,
Prior to introducing these applications in health care, a
Helium is inert and non-toxic. The last feature allows to
thorough fundamental study must be carried out to reveal
keep a good control of plasma chemistry, so that active
the reactions of objects so complex as cells and tissues. In
gases can be carefully dosed into the buffer gas.
this newly launched research, the choice of an adequate
The new plasma source has been characterized in terms
plasma source is crucial: it must operate under atmospheric
of basic physical properties. Molecular composition of the
pressure, be electrically and chemically safe and it may not
plasma has been studied using Raman spectroscopy. Under
cause any thermal damage to the living object. Since in
normal operating conditions (Helium flow of 2 l/min) the
most atmospheric discharges the gas temperature is too
air content due to diffusion from the outside air is below
1%. The voltage needed to operate the plasma is 200–400 V

e-mail: e.stoffels.adamowicz@tue.nl peak-to-peak; typical power consumption is below
# Physica Scripta 2004 Physica Scripta T107
80 E. Stoffels, R. E. J. Sladek and I. E. Kieft
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).
100 mW. The gas temperature has been determined using
optical emission spectroscopy. The rotational temperature
has been calculated from the rotational distribution within
the second positive group of N2 : This yields a rough
temperature estimation (300–350 K), but for treatment of
vulnerable living cells a much higher accuracy is needed.
Therefore, thermal load to the surface in contact with the
plasma has been monitored using a thermocouple. In Fig. 3
the temperature increase as a function of exposure time is
shown. At low power levels heating is tolerable: plasma
needle can be safely used to treat living objects, without
causing thermal damage. Exposure of skin, tooth and
gingiva to the plasma is entirely painless.
3. Bacterial inactivation using the plasma needle
Bacterial sterilisation using atmospheric plasmas is well
documented in the literature. Various types of discharges
have been applied: atmospheric glows [5], dielectric/
resistive barrier discharges [6] and coronas [7]. Sterilisation
can be achieved by direct treatment in the active plasma
zone, e.g., by introducing bacteria-containing aerosols into
the plasma [7]. This is particularly applicable for decon-
tamination of air in plasma-based air filters. In the active
plasma zone there are many factors that can cause bacterial
destruction: UV radiation with wavelength below 100 nm,
active radicals (species like O and OH) and charging of the
Fig. 2. The scheme of the experimental setup, showing the needle confined
coaxially in a plastic tube, the Helium supply and the radio-frequency
connection.
Physica Scripta T107
Fig. 3. Temperature recorded by a thermocouple as a function of time.
This is a Helium plasma with a gas flow of 2 l/min, the power input is
approximately 20 mW. A very small sensor has been used (thermal
capacity of 0.01 W/K), so that heating is relatively fast. Large samples are
heated much slower.
membrane of a bacteria. The latter process can lead to the
‘‘explosion’’ of the cell [8]. Alternatively, treatment can be
performed in a downstream mode [5,6], where the sample is
exposed to the afterglow. The remote zone contains only
radicals, but thanks to their activity decontamination is
still efficient. When large area plasma reactors are used
(with tens of Watts of power input), sterilisation is very
fast. The so-called D-value (the time of plasma exposure
needed to eliminate 90% of the original bacterial popula-
tion) lies often under 10 s. On the other hand, these values
may be influenced by the method of sample preparation.
Very often thin and somewhat dried films of bacteria are
treated. Under these conditions bacteria are more vulner-
able than in a thick agar matrix.
Gas discharges described above are very useful for
sterilisation of non-living surfaces (e.g., medical equip-
ment), but they are too aggressive to be applied in vivo. In
case of plasma needle, direct tissue exposure is possible.
Keeping in mind the future application in dentistry, we
have performed tests on bacterial inactivation while
keeping the bacteria under optimal conditions for growth.
Escherichia Coli have been used as a model system, because
they are easy to culture ex vivo and resistant to external
factors. 50 ml of bacterial suspension (a rather large droplet
with thickness of 2–3 mm) has been placed on an agar-
# Physica Scripta 2004

containing dish and treated with the plasma under the
mildest conditions (voltage of 190 V, distance needle to
droplet of 1 mm). Afterwards, the dishes have been
incubated and 24 to 48 h later the newly formed colonies
have been counted. Counting the colonies allows to verify
the ability to proliferate and survive on long term, so it
provides more useful information than observing indivi-
dual bacteria. Therefore, colony counting is the most
popular method of determining the bacterial activity. The
exact protocols can be found elsewhere [9]. In Fig. 4 a
typical survival curve is shown. The D-values deduced for
the plasma needle are of course higher than those for large,
high-power reactors: the time needed to inactivate 90% of
E. Coli is about 40 s. On the other hand, the low-power
needle is much more suitable for treatment of small dental
cavities, without heating and damage. Moreover, it causes
depth sterilisation of bacteria hidden in their normal
medium. The actual bacteria causing dental decay are
Streptococcus Mutans; they are much more vulnerable than
E. Coli, and therefore very difficult to be kept alive outside
the oral cavity. The experiments with E. Coli give
confidence that the needle will allow to inactivate the
Streptococcus. For practical purposes, 99% of the bacteria
in a dental cavity must be eliminated [10]; this corresponds
to a treatment time of 1–1.5 min. Afterwards, the cavity
opening should be sealed with a very small filling. As said
before, the treatment is painless.
In the next section it will be shown that mammalian cells
are much more resistant to plasma treatment than bacteria.
Thus, plasma treatment is a very promising method for in
vivo sterilisation, where bacteria are eliminated while the
body cells remain unharmed.
4. Plasma treatment of cells in culture
Plasma interactions with eukaryotic cells are much more
complicated than interactions with bacteria. The cells are
generally more resistant, and can have many interesting
responses of self-defence. Some of these responses are of
interest for refined, high-precision treatment of diseased
tissues. Therefore, we conduct a fundamental study in
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. 4. A bacterial survival curve as a function of duration of treatment
with the plasma needle.
# Physica Scripta 2004
Gas Plasma Effects on Living Cells 81
released. The latter causes an inflammatory reaction and
damage to the tissue, so it must be avoided in refined
treatment.
In our experiments we have used various types of cells:
epithelial-like fibroblasts of Chinese hamster ovary (CHO-
K1), mouse fibroblasts (3T3) and human epithelial cells of
lung carcinoma (MR65). These cells are good model
systems from the point of view of future applications:
skin treatment and wound healing.
Cells in culture usually form a two-dimensional sheet, as
shown in Fig. 5. They make contacts with their surround-
ings by means of trans-membrane proteins, called cell
adhesion molecules (CAMs): cadherins and integrins.
Cadherins connect the cells with each other, while integrins
attach the cells to the bottom.
During plasma treatment, cells are subjected to more
severe conditions than bacteria—voltages of at least 300 V
are needed to induce any reaction. The exposure time is 30–
60 s. The cells are covered by physiological saline solution,
to prevent them from desiccation and to divert static
electricity (the latter may cause membrane charging, as in
case of dry bacterial samples). The thickness of the liquid
film is 0.3–0.5 mm, the distance from the needle to the
liquid surface is 1 mm. After treatment, cells are observed
under a conventional phase-contrast or a confocal fluor-
escence microscope. Cell viability is assayed using fluor-
escent staining: propidium iodide (PI) to detect dead
(necrotic) cells and cell tracker green (CTG) to detect
living cells. The exact procedure is described elsewhere [11].
Generally, no necrosis is observed: cells remain alive after
plasma treatment. However, the interactions between them
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
spherical (see Fig. 6). Such detachment is initiated by the
plasma and it commences for about 20 s; under the
microscope it can be seen that the layer of cells ‘‘dissolves’’.
Cells remain in this state for several hours (2–6 h);
afterwards the cell contact is restored. We expect that
plasma species affect only the CAMs, while the cells remain
healthy and capable of replacing the damaged proteins.
This kind of plasma action can be seen as ‘‘surface
Fig. 5. A phase-contrast microscope picture of CHO-K1 cells, a control
sample. Cells are attached to the bottom and form a two dimensional
sheet.
Physica Scripta T107
82 E. Stoffels, R. E. J. Sladek and I. E. Kieft
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
high: the area of detached cells can be as small as 0.1 mm in
diameter. The borders between the ‘‘detached’’ and
uninfluenced areas are as sharp as a single cell thickness
ð20 mmÞ: Potentially, cell detachment is the finest way of cell
manipulation and it may become a minimum-destructive
method of disposing of unwanted cells.
The detailed mechanisms of bacterial decontamination
or cell detachment due to interactions with the plasma
needle are not yet completely clear. In both cases bacteria/
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
visible at the depth of 0.5 mm or smaller, while inactivation
of bacteria takes place in a layer that is millimetres thick.
Apparently the species, which cause the described effects
are able to penetrate under water. We expect that active
oxygen radicals (O, OH HO2 ; etc.) are responsible for
oxidating the CAMs, or poisoning the bacteria. Recently,
we have monitored active radicals in water samples, which
have been treated with the needle, and substantial
concentrations ð1–10 mmole=dm3 Þ have been found. The
radical measurements are beyond the scope of this paper
and will be reported in the near future.
5. Conclusions
At present, plasma techniques are being carefully intro-
duced in medicine. Several interesting and potentially
Physica Scripta T107
applicable effects have been identified, but the research is
still in the introductory/testing phase. Most of the studies
are of fundamental nature and they involve rather simple
model systems. However, the first results show that plasma
treatment has the potential of becoming a valuable method
in the surgery of the future. Bacterial decontamination
without harming the tissue, control of inflammation,
treatment of skin, wounds and caries are just a few
examples of possible applications. We are positive that
more sophisticated therapies are about to emerge.
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# Physica Scripta 2004