Superficial treatment of mammalian cells using plasma needle
E.Stoffels, I.E. Kieft, R.E.J. Sladek
Department of Biomedical Engineering
Eindhoven University of Technology
PO Box 513, 5600 MB Eindhoven, The Netherlands
e-mail: e.stoffels.adamowicz@tue.nl
Abstract
Interactions of a small-size, non-thermal helium plasma (plasma needle) with
living cells in culture are studied. We have demonstrated the non-destructive character
of our the plasma needle: under moderate conditions (low-power and low
concentration of molecular species) 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 Due to 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.
Introduction
Plasma processing of solid-state materials has more than twenty year long
history. Numerous techniques of surface modification have been investigated and
successfully implemented on the industrial scale: reactive ion etching of
semiconductor/photoresist, deposition of amorphous silicon for solar cells, production
of hard/protective/hydrophobic/decorative layers, etc. In all these applications, refined
treatment is obtained performed using non-thermal gas discharge plasmas. A nonthermal plasma is the only medium which combines These media combine
exceptional chemical activity with relatively mild, non-destructive character. All
processes occur at room or slightly elevated temperature, so that treated surfaces do
not undergo suffer any thermal damage. Thanks to the latter feature, plasma is suitable
for treatment of heat-sensitive organic materials, like plastics and fabrics (wool [1]).
Plasma application in life sciences is a recent trend, naturally born from the
well-established and thriving solid-state processing technology. As expected in such a
new development, many obstructions have been encountered. For example, most nonthermal discharges are generated under reduced pressures (< 1 mbar), which limits the
choice of materials suitable for plasma processing, but not all organic materials can
withstand such conditions. Nevertheless, low-pressure plasmas are being successfully
used in biomedical engineering, e.g. for surface patterning to control cell adhesion [2]
or spraying of bio-compatible materials to improve the performance of artificial
implants [3]. Intermediate pressure (1- 10 mbar) and sub-atmospheric discharges
have proven to be efficient in bacterial decontamination [4]. Many plasma-based
devices have been constructed to sterilise (plastic) medical equipment; air filters have
been equipped with corona-like discharges to destroy bacteria and spores in public
areas [5].
Non-thermal plasmas at low pressures are undeniably of high value in surface
biomedical technology. However, it is much more convenient to operate plasma
devices at ambient pressure. Atmospheric plasmas are more flexible and less
expensive in operation, because they do not require costly vacuum systems. Although
obtaining non-thermal plasmas under atmospheric pressure is not an easy task, there
are already several sources available [6-8]. Not all of them are directly applicable for
treatment of vulnerable organic materials, but they can be used as light sources or
sterilising, cleaning and etching media.
We have developed an atmospheric plasma (the plasma needle), which
operates at room temperature [9]. The physical principle of obtaining such a nonthermal source is maximisation of its surface to volume ratio: electron-induced
heating in the volume becomes negligible, while energy losses through the plasma
surface are relatively high. This situation is common in micro-plasmas, where the size
of the glow is below 1 mm. Plasma needle is an example of such a small atmospheric
glow. Its small size guarantees a low gas temperature, and in addition it allows for a
high-precision local surface treatment. Measurements of temperature at the processed
surface will be presented further in this paper. Thermal effects during treatment with
the plasma needle will be discussed further in this paper.
Plasma needle will in future be used as may become a refined surgical tool to
dispose of pathological cells (cancer) and unwanted tissues (peeling, removal of
scars), to fight bacterial infections (in vivo sterilisation of skin and dental cavities) and
to improve wound healing by controlling cell adhesion. In contrast to mechanical,
thermal or laser methods, plasma treatment will not cause severe injury and cell death
(necrosis). Mechanical, thermal or laser methods, often used in surgery, always cause
severe cell injury and death (necrosis). In the latter process cell membrane is damaged
and the released cytoplasm induces an inflammatory reaction in the tissue. In contrast,
necrosis can be avoided in plasma treatment, and cell removal without inflammation
can be performed. In this study we investigate and classify the possible ways in which
the plasma can affect mammalian cells. We use two model systems: the Chinese
hamster ovarian cells (CHO-K1) and the human cells of lung carcinoma MR65. The
CHO-K1 cells are basal type (fibroblasts), used as a first model to identify general cell
responses, while MR65, being human epithelial cells, bring us closer to the intended
medical application (skin treatment). We have established that plasma treatment
causes no necrosis, but a sophisticated cell response: temporary interruption of cell
adhesion.
Experiment
The plasma needle
Plasma needle is a radio-frequency (rf) glow generated at the tip of a sharp
tungsten needle (length 5 cm, thickness 0.3 mm) contained in a metal/plastic plasma
box. The discharge is generated using a Hewlett Packard 33120A waveform generator
in combination with an Amplifier Research 75AP250RF amplifier and a home-built
-type matching network. The excitation frequency is about 13 MHz and peak-topeak RF voltage is from 200 to 400 V. Plasma power is determined using Amplifier
Research PM 2002 power meter connected to an Amplifier Research dual directional
coupler. A schematic view of the setup is shown in Figure 1. Basically, this is a
unipolar configuration, where (remote) chamber walls serve as a grounded electrode.
Similar to other atmospheric glows, the plasma operates most readily in helium.
However, this is not a hindrance: for the safety of treated tissues it is favourable to use
an inert gas as an ambient atmosphere and allow only small amounts of active species
(e.g. air). Helium supply into the plasma box is controlled by a Brooks series 5850E
mass flow controller. The box is not vacuum tight; under typical operating conditions
(He flow of 2 slm) the air content in the plasma due to leakage from outside
atmosphere is 0.5 to 1%. The plasma box is supplied with two external manipulators:
one to move the stage on which treated samples are placed, and another one to adjust
the distance between the needle and the sample surface.
Diagnostics
In this multi-disciplinary research we combine physical methods to
characterise the plasma with standard assays from cell biology to recognise the
condition of the treated cells. In the past we performed optical emission spectroscopy
to determine various temperatures in the plasma (vibrational, rotational and electronexcitation [9]). Here we present temperature measurements using a common NiCr-Ni
thermocouple, fixed to the moving stage. No influence of the rf noise on the
thermocouple read-out has been observed.
Chinese Hamster Ovarian (CHO-K1) and MR65 cells are cultured in flasks
containing appropriate cell culture medium, and incubated at 37 oC. The exact
protocol is given elsewhere [10]. To prepare samples for plasma treatment, the cells
are transferred onto sterilized object glasses (26 x 10 x 1 mm) and placed in multiwell dishes. Healthy cells proliferate once every 24 hours and form a twodimensional layer on the glass sample (see Figure 2). We typically wait for two or
three days before exposing them to the plasma. This is in order to obtain an optimum
confluence (the percentage of the cell-covered area on the glass sample), which is
about 80%. Just before treatment, samples are washed with Phosphate Buffered
Saline (PBS) and placed on the moving stage. To prevent drying out of the cells,
samples are covered with a film of PBS (2 droplets or 0.08 ml; the resulting thickness
of the PBS layer is about 0.3 mm). Plasma needle is brought at a distance of 2 mm to
the sample. Typical treatment time is 30 seconds, during which the sample is moved
by the manipulated stage over a typical distance of 1 cm. This produces a typical
"track" of plasma-treated cells, which can be easily recognised under the microscope.
The track is typically 0.5 mm wide. Individual cells on this track are irradiated for
about 1-2 seconds.
In order to establish the condition of cells after treatment, appropriate
fluorescent staining is applied. For observation, a confocal laser scanning microscope
(CLSM) is used. The CLSM is equipped with an argon ion laser (488 nm), which
excites the fluorescent probes applied to the cells. The resulting fluorescent radiation
is focused on the pinhole (see Figure 3) in front of the detector supplied with an
appropriate colour filter. Note that in this configuration the fluorescent light collected
by the detector originates mainly from the focal spot of the laser; light emitted from
other areas is greatly suppressed. In biophysics confocal microscopy is a standard
technique, allowing for three-dimensional imaging with a spatial resolution of 0.2
m. Typically we use two fluorescent probes: Cell Tracker Green (CTG) and
Propidium Iodide (PI). CTG is absorbed by all cells, but only living cells transform it
into fluorescent species. This probe is used to verify cell viability: in living cells the
whole cytoplasm displays green fluorescence. PI penetrates only necrotic cells (with
damaged membranes) and binds to the DNA and RNA. So-called dual staining
(CTG+PI) is applied about 1 hour after plasma treatment, in order to distinguish
between dead and living cells.
Results and discussion
Plasma appearance and interactions with surfaces
In the unipolar configuration, when grounded objects are remote, the plasma
appears exclusively at the tip of the rf-powered needle. Dependent on the conditions,
the glow can assume various shapes; some examples are shown in Figure 4. This is a
spatially self-constricted plasma. The size of the glow is determined by the power
input and the composition of the ambient gas. The peak-to-peak rf voltage at the
breakdown is about 200 V; the plasma appears as a faint point-like glow. At about
350 V it starts to expand in volume, spreading along the exposed part of the powered
wire. In presence of electron attaching species (oxygen due to air leakage) the glow
shrinks into a narrow flame. The breakdown voltage increases to about 400 V for 10%
air contamination; the plasma becomes sensitive for arcing and instabilities. Previous
measurements have shown that both high power level and presence of
(electronegative) molecular species result in considerable heating of the gas, even up
to 600 K [9]. For the treatment of living cells we apply power levels not higher than
0.2 W and we limit air concentration to less than 1% of the ambient atmosphere.
When the plasma needle is brought close enough to a (grounded) object, it
switches into a bipolar mode, where the glow spreads towards the surface (see Figure
4c). The threshold distance is dependent on the power level; for 0.2 W it is about 2
mm. In this way the plasma is able to interact with the living objects, placed on the
grounded sample holder. In order to verify that the objects (cells) do not suffer
thermal damage, we have measured the surface temperature by means of a
thermocouple attached to the sample holder. Two configurations are tested: a dry
thermocouple (metal surface) and a thermocouple immersed in PBS (liquid surface).
The PBS film is very thin; the distance between the thermocouple bead and the
plasma needle is in both cases the same. Temperature measurements in PBS are
relevant for cell treatment and also for in vivo plasma operation. This is because living
cells and tissues are vulnerable to desiccation, so they must be covered by a film of
ionic solution. The steady-state temperature as a function of the distance between the
surface and the needle is shown in Figure 5. It is evident that the thermal effect is
directly related to the plasma power. At low power levels the temperature increase is
minor and thus tolerable for in vivo treatment.
Cell treatment
Normally, cultured cells form a single layer on the substrate. This kind of twodimensional tissue tends to fill the whole available surface. When the substrate is fully
occupied (100% confluence), cell proliferation is reduced and eventually the cells die.
Therefore the unfixed (living) samples cannot be cultured longer than about 4 days.
Formation of a sheet consisting of elongated cells results from cell-cell and cellbottom interactions by means of trans-membrane proteins, called cell adhesion
molecules (CAM). Here, two kinds CAMs are active: cadherins, which bind
neighbouring cells to each other, and integrins, which attach the cells to the substrate
surface.
Treatment of CHO-K1 and MR65 cells is performed under mild conditions:
plasma power not exceeding 0.1 W and exposure time of about 1 second. Applying
higher power levels results in necrosis of the cells, which are at the nearest distance to
the plasma source. Typical necrotic cells stained with PI are shown in Figure 6. The
cells seem to retain their internal structure (nuclei, etc.) but their membranes are
leaking. We suppose that the membranes are etched by active radical species from the
plasma. In pursuit of fine, non-destructive plasma treatment, inducing cell necrosis
should be avoided.
The most striking effect of plasma treatment is the interruption of cell
interactions. Typical treated areas in CHO-K1 and in MR65 cell samples are shown in
Figure 7. When the cells are detached from each other, they assume a round shape
(optimal surface to volume ratio). Cell detachment after exposure to plasma seems to
be a general feature, which takes place for various cell types. The viability of the
detached cells has been confirmed using CTG+PI. Rounded cells display intense
green fluorescence; on the same sample no necrotic cells can be found. Later on, the
rounded cells have been fixed (devitalised) using buffered 4% formaldehyde, and
stained with PI in order to visualise possible modifications to the cell interior (e.g. to
the DNA in the nuclei). However, no remarkable changes have been observed. This
shows that plasma action is restricted to the cell exterior. The unfixed rounded cells
restore cell contact within 4 hours; after 24 hours the sample is confluent, just like any
non-treated sample. At somewhat longer plasma exposure, the cells not only detach
from each other, but also lose contact with the substrate and float in the PBS. This
results in the formation of typical “voids” on the sample surface (Figure 8). The fully
detached cells can be taken up in the medium and transferred onto another substrate.
Also in this case, CHO-K1 as well as MR65 cells are viable and tend to reconstruct a
layer within a few hours. We expect that this kind of cell behaviour is induced by
plasma particles, which can penetrate under water. A “suspected” class of species are
the reactive nitrogen and reactive oxygen species (RNS/ROS). These reactive species
include oxygen atoms (O), oxygen negative ions (O2-), ozone (O3), hydroxyl radicals
(OH), nitric oxide radicals (NO) and hydrogen peroxide (H2O2). The observation that
the effect of detachment becomes more pronounced with increasing air contamination,
but is fully eliminated when the plasma is applied through a layer of glass (e.g. when
the samples are put upside down on the stage), indicates that ROS/RNS play a role in
cell detachment. Since the radicals can propagate under liquid only to a limited extent,
their densities will rapidly decrease with increasing penetration depth. This situation
is schematically depicted in Figure 9. Based on strong density gradients, it can be
explained that loss of cell-cell intraction is easier to attain than the total detachment
from the substrate. Since the cells are viable, the damaged CAMs are reconstructed
within a few hours, which is a typical time scale for protein synthesis in healthy cells.
In the near future we will apply a fluorescent probe to detect ROS in
(biological) liquids. This will allow to determine the concentration gradients and also
penetration of active oxygen species into the cell interior.
Conclusions
Plasma interactions with living cells do not necessarily cause cell death. In
fact, we have found a non-destructive plasma treatment which can be considered as
“surface processing” of cells. We observe loss of cell contact, and cell detachment
from the substrate, possibly due to plasma-induced damage of cell adhesion
molecules: cadherins and integrins. Such a situation is temporary; a full restoration of
cell interactions is completed within a few hours. This remarkable plasma-cell
interaction allows ample time to manipulate the detached cells: they can be removed,
rearranged or transferred onto other samples. Since the treated cells do not seem to be
harmed, we suppose that cell detachment is the finest and least destructive action of
the plasma.
The final aim of this research is achieving controlled tissue modification with
a high precision. The plasma effect on cell adhesion is potentially applicable in
refined cell removal. Since the cells that detach during interaction with the plasma are
alive (not necrotic), no inflammatory reaction in the tissue is expected. Besides
simple cell removal (e.g. disposing of pathological cells), plasma treatment offers a
possibility to aid wound healing, by making cells move into the injured area. Due to
its mild and refined action, plasma may prove advantageous in various therapies,
where minimum damage is of high importance.
References
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Figure captions
Figure 1: A scheme of the experimental setup. The details of the plasma box: a – stage
manipulators, b – the needle (rf electrode), c – the sample, d – the thermocouple head.
Figure 2: A culture of healthy CHO-K1 cells. The cells are stained with cell tracker
green and visualised by the confocal fluorescence microscope. Average length of a
cell is 30 m.
Figure 3: A scheme of a confocal microscope. The sample is placed on the stage
(bottom of the picture) and irradiated by a laser. The induced fluorescence/scattering
is led through a system of two lenses and finally detected by the photomultiplier (for
more details see http://www.zeiss.com).
Figure 4: Various shapes of the plasma glow: (a) 1 mm monopolar glow in helium at
250 V (about 0.1 W), (b) expanded glow at 400 V (about 1 W), (c) bipolar discharge
created between the needle and a moist organic tissue.
Figure 5: Surface temperature determined by a thermocouple as a function of distance
of the needle to the surface, for various conditions: dry thermocouple: circles - 0.3 W
plasma in helium, squares - 0.15 W in helium, triangles - 0.15 W in helium with 3%
air; thermocouple immersed in PBS: diamonds - 0.15 W in helium.
Figure 6: Necrotic (dead) CHO-K1 cells after treatment at 1 W. The cell membranes
are damaged; the nuclei display bright red fluorescence after PI staining.
Figure 7: Plasma-treated (0.1 W) samples of CHO-K1 cells (left) and MR65 (right).
CHO-K1 are stained with CTG; the green fluorescence confirms their viability. MR65
are observed under a usual phase-contrast microscope (without staining).
Figure 8: Overview of the treated area on a CHO-K1 sample: the typical void (0.5 mm
in diameter) is formed due to local detachment of the cells from the sample. Partially
detached (rounded) cells are visible at the edge of the void.
Figure 9: A scheme depicting the possible scenario of plasma-cell interactions leading
to cell detachment.
Figure 1. Stoffels et al.
function
generator
rf amplifier
dual coupler
Power
meter
He bottle
a
flow controller
b
c
a
d
matching
network
Figure 2. Stoffels et al.
Figure 3. Stoffels et al.
Figure 4. Stoffels et al.
a
c
b
Figure 5. Stoffels et al.
temperature
30
0.3 W
28
26
24
0.15 W
22
1
3
5
7
distance to needle (mm)
9
Figure 6. Stoffels et al.
Figure 7. Stoffels et al.
Figure 8. Stoffels et al.
Figure 9. Stoffels et al.
cadherin
integrin
plasma
helium
solution
radical density gradient
fully detached cell
attached cells