st
21 International Symposium on Plasma Chemistry (ISPC 21)
Sunday 4 August – Friday 9 August 2013
Cairns Convention Centre, Queensland, Australia
Non-Thermal Atmospheric Pressure Plasma Treatment of Human Cells: The
Effect of Ambient Conditions
A. Barton1, K. Wende1, L. Bundscherer1, K.-D. Weltmann2, U. Lindequist3, K. Masur1
1
Centre for Innovation Competence plasmatis, Greifswald, Germany
2
Leibniz Institute for Plasma Science and Technology (INP Greifswald), Greifswald, Germany
3
Institute of Pharmacy of the Ernst Moritz Arndt University of Greifswald, Greifswald, Germany
Abstract: The ambient conditions of non-thermal atmospheric pressure plasma were modified
by admixtures of O2 and N2. It was analyzed how the ambience can influence the cell viability
of two wound healing related cell lines (keratinocytes HaCaT, monocytes THP-1) after plasma
treatment. The ambient conditions have significant effects on cytotoxicity and the cell lines
seem to react with different defense mechanisms.
Keywords: plasma medicine, HaCaT keratinocytes, THP-1 monocytes, cell viability, kinpen
1. Introduction
Non-thermal atmospheric pressure plasma is a promising tool for the treatment of chronic wounds. ROS and
RNS are believed to play a central role in biomedical applications of plasma [1]. In order to obtain a deeper insight into the underlying mechanisms, the argon plasma
treatments were conducted under different controlled ambient conditions, varying the nitrogen to oxygen ration of
the ambient gas. While it is well known that effects of
plasma treatment on apoptosis and proliferation of human
skin and immune cells are dose dependent [2, 3], we
found that their viability can be regulated by controlling
the ambient plasma conditions [4]. Our results show the
dose dependent impact of non-thermal plasma on the investigated keratinocyte (HaCaT) and monocyte (THP-1)
cell lines and give first insights into the importance of
controlled ambient conditions for plasma treatments on
cells.
2. Experimental
The plasma treatments were performed with the atmospheric pressure plasma jet kinpen [5] (Neoplas, Germany) in combination with a shielding device which was
explained in detail by Reuter et al. [4] and shown in figure
1. The kinpen was operated with argon (3 sLm) and the
curtain gas consisted of different ratios of O2 and N2 with
a total volumetric flow rate of 5 sLm. The oxygen to nitrogen ratio in the shielding gas was varied in five steps
from 0 % O2 and 100 % N2 to 100 % O2 and 0 % N2 with
25 % step size.
The human keratinocyte cell line HaCaT (DKFZ, Germany) and the human monocyte cell line THP-1 (CLS
Cell Lines Service, Germany) were used for cell vitality
experiments. Every three to four days both cell lines were
subcultivated. Keratinocytes were passaged 1.3 x 106 cells
mL-1 and monocytes 0.2 x 106 cells mL-1, in Roswell Park
Memorial Institute (RPMI) 1640 medium (Lonza, Switzerland) supplemented with 8 % (HaCaT) or 10 %
(THP-1) fetal calf serum (Sigma-Aldrich, United States of
America), 2 mM L-glutamine and antibiotics (0.1 mg L-1
streptomycin and 100 U mL-1 penicillin) (Lonza, Switzerland).
Fig.1
Scheme of the plasma treatment of cell culture medium.
The atmospheric pressure plasma jet kinpen was operated with
argon and the shielding device with nitrogen and oxygen.
The analysis of cell viability was performed with the
CellTox™ Green Cytotoxicity Assay (Promega, United
States of America). The CellTox™ Green Dye is not cell
permeable and stains intracellular DNA after cell membrane becomes leaky due to cell death. To measure the
cell viability after plasma treatment with different gas
shieldings 15,000 keratinocytes in 75 µL cell medium
st
21 International Symposium on Plasma Chemistry (ISPC 21)
Sunday 4 August – Friday 9 August 2013
Cairns Convention Centre, Queensland, Australia
were seeded per well for attachment in a 96 well-plate 24
hours before. The plasma treatment of the cells was performed in an indirect way, which means 5 mL of medium
were treated. Subsequently the cell medium was replaced
by 75 µL treated medium. The non-adherent Monocytes
grow in suspension, therefore the protocol had to be
adapted accordingly. First, they were harvested and
counted with the Cedex XS Cell Counting System (Roche,
Germany) and subsequently resuspended with the plasma
treated medium (15,000 cells/ 75 µL). At least 75 µL of
the treated cell suspension were transferred per well into a
96 well-plate.
In addition, cells treated with 100 µM H2O2 as well as
untreated cells were conducted for both cell lines. After
incubation time of 24 hours the CellTox™ Green Dye was
added to the cells and incubated for 15 min. Afterwards
the fluorescence was measured with the microplate reader
Infinite 200 PRO (Tecan, Switzerland) with the settings
appropriate. At least six replicates were measured for all
samples.
3. Results and Discussion
Both cell lines displayed a dose dependent behavior after plasma treatment. The longer the plasma exposure the
higher the cytotoxicity signals. The keratinocytes were
plasma treated for 20 and 180 s in combination of five
different mixtures of ambient gases as presented in figure
2. For every ambient condition it was demonstrated that
the cytotoxicity was significantly higher for 180 s than for
20 s plasma treatments. Interestingly, the ratio of oxygen
to nitrogen in the ambience of plasma seemed to play an
important role for cell response. Keratinocytes which
were plasma treated for 20 s showed a slight increase of
cell death from 0 % to 75 % oxygen in the shielding gas
peaking at 100 %. For 180 s the increase of oxygen in the
ambient gas from 0 to 100 % induced a significantly increased cytotoxicity.
The monocyte cell line THP-1 was treated with plasma
for 20 and 180 s and H2O2 (100 µM). Due to the fact that
the immune cells did not show any changes compared to
untreated cells, cells were treated in an additionally experiment with 200 µM H2O2 and for 360 s with plasma.
Figure 3 just illustrates the significant cell responses after
360 s plasma treatments with a cytotoxicity maximum at
75 % oxygen and 25 % nitrogen in the shielding gas. The
condition with 100 % oxygen to 0 % nitrogen reduced
cytotoxic response.
Although the core plasma was not changed and only the
ambient gas mixtures varied, the two investigated human
cell lines revealed different cellular reactions after plasma
treatments. The keratinocytes HaCaT increased the cell
death with enhanced oxygen amount in the shielding gas
whereas the monocytes THP-1 showed a maximum of
dead cells at a surrounding gas mixture of 75 % O2 to
25 % N2. This progression of the immune cells could be
Fig.2
The keratinocyte cell line HaCaT was indirect treated
with plasma (20 s, 180 s) and hydrogen peroxide. The plasma
treatment was done with five ambient conditions. All
flourescence signals were normalized to the untreated control
cells. Statistical analysis was done via one-way ANOVA (prism
6.0 graph pad software) (*, p < 0.05).
explained by the fact that monocytes produce reactive
species themselves to degrade phagocytosed microorganisms. Due to this they also generate a relatively high
amount of antioxidant enzymes like catalase, thioredoxin
reductase and glutathione peroxidase for self-protection
[2]. This is probably the reason why the monocyte cell
line THP-1 revealed normal viability after 180 s plasma
treatment and only initiated cell death after 360 s plasma
exposure.
Fig.3
The monocyte cell line THP-1 was indirect treated with
plasma (360 s) and hydrogen peroxide. The plasma treatment
was done with five ambient conditions. All flourescence signals
were normalized to the untreated control cells. Statistical analysis was done via one-way ANOVA (prism 6.0 graph pad software) (***, p < 0.001).
st
21 International Symposium on Plasma Chemistry (ISPC 21)
Sunday 4 August – Friday 9 August 2013
Cairns Convention Centre, Queensland, Australia
As a reason for the steady rise of the keratinocytes cell
death in correlation with rising oxygen concentration it
could be hypothesized that the capacity of the oxidative
stress defense mechanisms of the HaCaT cell line is limited. With increasing oxygen amount in the shielding gas
and expectable rising ROS production like O or O3 in the
gas phase, the cells need more defense mechanisms to
protect themselves from the rising oxidative stress. If these protection possibilities reach a limit the skin cells
eventually died.
In case of a gas shielding of pure oxygen or pure nitrogen, no reactive oxygen-nitrogen species (RONS) like NO,
NO2 or HNO3 can be produced in the gas phase. From the
behavior of the monocyte cell line it therefore could be
inferred that they do have better protection mechanisms
for ROS or RNS than RONS, because plasma treatments
with a higher amount of RONS (75 % O2/ 25 % N2) were
more cytotoxic than treatments with only RNS or ROS
(100% O2 or 100 % N2). The monocytes express for example the antioxidant enzyme catalase in a high copy
number, an enzyme which decomposes the reactive oxygen species hydrogen peroxide.
Schmidt, S. Bekeschus, S. Hasse, K.D. Weltmann, K.
4. Conclusion
These results indicate an important influence of the
ambient conditions on cells during plasma treatment. Both
investigated cell lines HaCaT and THP-1 reveal significant changes for cell viability after treatments with different ambient gas mixtures. The cells also seem to have
different abilities to deal with ROS, RNS or RONS. All in
all, pure nitrogen in the shielding gas showed the lowest
cytotoxicity for both cell lines, whereas oxygen dominated shielding showed the highest influence on the investigated cell lines.
For biomedical applications plasma sources in a controlled ambient are a very promising tool because they
enable the regulation of cellular responses in a defined
way.
[5] K.D. Weltmann, E. Kindel, R. Brandenburg, C. Meyer,
5. Acknowledgment
This work is funded by German Federal Ministry of
Education and Research (grant number 03Z2DN11).
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