22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Effects of air DBDs on eukaryotic cells and biological liquids
I. Trizio1, R. Gristina2, E. Sardella2, E. Francioso3, G. Dilecce2, M. Schmidt4, Th. von Woedtke4 and P. Favia1,2
Department of Chemistry, University of Bari ʺAldo Moroʺ, Bari, Italy
Istitute for Inorganic Metodologies and Plasmas(IMIP)-CNR, Bari, Italy
3
Veterinary Clinics Section (D.E.T.O.), Bari, Italy
4
INP-Leibniz Institute of Plasma Science and Technology, Greifswald, Germany
1
2
Abstract: In the present work the selective interaction between a dielectric barrier
discharge and two different types of eukaryotic cells has been evaluated. A dose-dependent
response in both type of cells has been found. Moreover plasma-induced changes in
biological liquids enhanced the cell growth of stem cells.
Keywords: dielectric barrier discharge, eukaryotic cells, reactive species
1. Introduction
Over the last two decades non-equilibrium atmosphericpressure (AP) plasmas are routinely used in material
processing applications [1]. In recent years AP plasmas
are being also widely investigated for surface engineering
of biomaterials [2]. AP plasmas entered in this field
because of their mild conditions, their ability to ignite
plasmas in small volumes and the limited use of vacuum
systems [3]. Among these attractive features, the potential
of to achieve high reactivity in the gas-phase and on
surfaces without significant increase of temperature
opened up more recently the possibility of using airplasma treatments directly on heat sensitive materials
including cells and biological tissues. This novel
approach, resulted in a burst of research activities on
tumour and skin disease treatments, as well as in dental
and tissue engineering applications just to mention a few
[4-6]. Although until today a complete understanding has
still to emerge on the application of this technology on
eukaryotic cells, it is clear that the amount of each
different component of plasma (reactive species, electric
field, radiations, ions, charged particles, etc) has to be
carefully dosed to properly tune the changes in biological
response stimulated by the plasma exposure [5]. Up to
now an exact definition of the ‘plasma dose’ is still an
open question due to the complex interconnections of
plasma components and parameters; on the other hand,
though, it is believed that low doses of plasma are able to
stimulate proliferation of cells while higher doses induces
severe cytotoxic effects, inhibit cell proliferation and can
lead to cell apoptosis. This is clearly interesting when
applied to a wide range of tumoural cells including
melanoma, breast cancer cells and hepatocellular
carcinoma [6,9,10]. The plasma-cell interaction depend
on the way the plasma is generated, as well as on the way
of delivery and the organism to which is applied. To get
more insights on this, for the present research non-thermal
plasma has been used in two different approaches: Cell
Plasma Treatment (CPT) and Medium Plasma Treatment
(MPT). The first approach aimed in studying the effect on
cells of the plasma itself, consisted in direct exposure to
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plasma of two different types of eukaryotic cells, a Saos-2
human osteoblast line and Bone Marrow Stem Cell
(BMSC) primary line. The second approach consisting in
the only exposure of the culture medium to plasma, has
been followed to evaluate the role mediated by the active
species produced on treated medium on the cell
behaviour. It is known that this kind of treatments
exposes the sample to short and long-lived neutrals atoms
and molecules, including ozone (O 3 ), nitrogen monoxide
(NO), hydroxyl radicals (OH) and singlet oxygen (O 2 1Δ g )
[6,7]. But it is thought that more than the other plasma
agents, the reactive oxygen and nitrogen species (RONS)
generated by plasma could be the main causes of these
effects [11-13]. The Dielectric Barrier Discharge (DBD)
equipment used in this research has been developed in
collaboration with researchers of Leibniz Institute of
Plasma Science and Technology (INP, Greifswald, GER).
In this paper, it is shown not only the dose-dependent cell
response and the selectivity between two cell types but
also that the main tailoring plasma effect on cell
behaviour is mediated by changes in the medium.
2. Materials and methods
The DBD system utilized for this study is shown in
Fig.1 A) and B). It was based on a volume discharge
produced by a parallel electrodes geometry. The high
voltage (HV) copper plate electrode was covered with a
1mm thick glass dielectric while the ground stainless steel
mesh was spaced by the dielectric by a 1mm thick Teflon
ring. In Fig.1 C) the electric scheme of the equipment is
reported. The voltage, applied to the electrodes at 6KHz
frequency in pulsed mode, was measured by means of a
high voltage probe (Tektronix P6915A), while the current
was evaluated by measuring with an oscilloscope
(Tektronix TDS 2014C), the voltage drop across the
100nF capacitor connected in series with the ground
electrode. To treat cells or medium, the electrode system
was installed on the top 60 mm diameter cell culture. The
pulsed ambient air plasma was generated between the
two electrodes in close vicinity and without any contact
with cells or medium to allow the plasma active species
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diffuse through the stainless steel mesh toward the
sample.
Fig. 1. a) Picture and b) electrical scheme of the DBD
system based on copper HV electrode, a glass dielectric
and a stainless steel mesh separated by a Teflon spacer; c)
electrical scheme of the equipment.
In Table 1. the three different experimental conditions
utilized in this work are reported, listed from the milder to
the harsher one.
Table 1. Experimental parameters.
Condition
Mild (1)
Medium (2)
Harsh (3)
Voltage ( KV)
13
13
Frequency (KHz)
6
6
6
100
100
100
25(75)
Period (ms)
ton (toff) (ms)
13
10 (90)
25(75)
Dissipated Energy (J)
4
11
23
Energy dose (J/cm2)
0.2
0.6
1.2
Time of treatment (s)
15
15
30
The experiments have been performed with the human
Saos-2 osteoblast cell line (ICLC) and with sheep BMSC
cells. Both kind of cells were harvested in Dulbecco’s
Modified Eagle Medium (DMEM, Sigma Chemical Co.),
supplemented with 10% heat-inactivated fetal bovine
serum (FBS), 50IU/ml penicillin, 50 IU/ml streptomycin
and 200 mM glutamine and maintained at 37°C in a
saturated humid atmosphere of 95% air and 5% CO 2 .
Cells were detached from the flask with a trypsin/EDTA
solution (Sigma), and suspended in DMEM at a
concentration of about 1.7x104 cell/ml; the Petri dishes to
be treated were seeded with 3 ml of this cell suspension
(CPT) or 3ml of DMEM (MPT) .
Before the CPT or MPT, cells were left 24h in the
incubator to allow adhesion to the bottom of the Petri
dish, then the medium was aspired. Wet cells were soon
exposed to the plasma, and DMEM was added soon after.
The cells growth was stopped at 24, 72 and 144h after the
2
CPT or at 23, 71 and 143h after being in contact for 1h
with plasma treated DMEM (MPT).
The mitochondrial activity of the cells, representative of
their viability was determined with the MTT colorimetric
assay. Such assay is The cell growth was stopped at 24h,
72h and 144h after the plasma treatment. The optical
density (OD) related to the Formazan production in living
cells was measured with a JENWAY 6505 UV/Vis
spectrophotometer at 570nm with respect the reference at
690nm. For assessing the cell morphology 24, 72 and
144h after each treatment, cells were fixed in 4%
formaldehyde/PBS solution (room temperature, 20min)
and stained in a dye solution of Coomassie Brilliant Blue
R250 (Sigma; for 3min in 50% methanol, 10% acetic
acid). Dyed cells were observed at different
magnifications with a phase contrast Leica DM ILI
microscope. Statistical analysis was performed using
Two-way ANOVA followed by Bonferroni Post-Test.
Differences were considered statistically significant for
p<0,05.
3. Experimental results
In Fig.2A) the viability of Saos-2 cells 144h after from
the CPT and MPT is compared for the three exposure
conditions reported in Table 1. It appears clearly that, in
both CPT e MPT the response of Saos-2 cells is strongly
influenced by the conditions of the treatments, i.e. the
harsher was the conditions (higher dissipated energy and
energy density; longer t on and treatment time), the lower
the viability of Saos-2 cells after 144h of culture.
Moreover, the evidence that the MPT exposure induced a
cell response very much similar to that induced in the
CPT leads to think that the main effect of plasma on the
cells is mediated by reactive species in culture medium. It
is very interesting to note that the Mild condition in the
MPT resulted in a viability comparable to that of the
control (p<0.05). In Fig. 2B) the viability of BMSC cells
is measured after 144h from the CPT and MPT in the
three conditions. Extremely interesting results have been
obtained in this case, compared with the results shown in
Fig. 2A). Both CPT and MPT in the three conditions, in
fact have produced less effects on the growth of stem cells
with respect to the Saos-2. In particular it can be observed
that the treatment performed on medium (MPT) in the
Mild and Medium Conditions have activated a significant
increase of the BMSC cell viability with respect to the
control, very differently from the CPT the behaviour of
the Saos-2 cells.
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favorable growth conditions. After the three treatments,
on medium only, instead, Saos-2 cells were dreastically
reduced in density, and shown lower clusterization and
different morphology with respect to the control. After the
Harsh condition the few remaining cells appeared single,
spherical or spindle-shaped and not organized in cluster.
The same assay has been performed also for BMSC cells,
characterized by a different behavior and organization
with respect to the Saos-2 as visible in the control. In
agreement with the increased viability data shown in Fig.
2B, BMSC cells treated with the Mild condition of the
MPT, exhibited a total confluence with respect to the
control, confirming higher proliferation activated by the
medium plasma treated in the Mild Condition.
Fig. 3. Saos-2 and BMSC stained with Coomassie blue.
Cells have been grown for 144h after indirect plasma
treatment in three different experimental conditions.
In order to match the dose-dependent cell response with
plasma-induced changes in treated liquid, the ROS and
RNS detection based on wet-chemical reaction has been
performed. To this purpose various liquids have been
tested starting from water, considered as model liquid,
phosphate buffer solution (PBS) and DMEM culture
medium.
Fig.2. A)MTT viability results of Saos-2 cells after 144h
from the CPT and the MPT; B) Viability of BMSC cells.
1) stands for Mild condition, 2) for Medium condition
and 3) for Harsh condition). Statistical differences
between means were calculated with Two way ANOVA
and Bonferroni Post Test.(=:p<0.05 vs Control; +:
p<0.05 vs CPT1; /:p<0.05 vs CPT2; ):p<0.05 vs CPT3;
!:p<0.05 vs MPT1; ?:p<0.05 vs MPT2; $:p<0.05 vs
MPT3).
Fig. 3 shows the control and MP treated Saos-2 and
BMSC cells after staining with Coomassie Blue, after
144h of culture. It is interesting to note that these
observations confirm the MTT viability data reported
above. Staining data after CPT (not shown) are also in
agreement with the corresponding viability data shown in
Fig. 2. In Fig.3 control Saos-2 cells after 144h exhibit
their morphology and cluster organization typical of
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4. Conclusions
The data shown in this paper seems to confirm that low
doses of AP air-plasmas are able to activate cell
proliferation, at least in our Mild MPT mode, and induce
a selective response on different cell lines. As reported in
literature, this opens the possibility of using plasma
technology for killing cells (cancer cells, bacteria) at one
hand and, at the other hand, for activating cell
proliferation of primary stem cells. Plasma-activation of
cell proliferation is already utilized in vivo for wound
healing applications, but can become a useful approach
also in tissue engineering and regenerative medicine.
Deeper investigations are clearly needed in the field to
sharply define the density of all active species generated
and diffused in water, buffers and biological media as a
function of the plasma exposure parameters, and to fully
understand the interaction of each kind of active species
with various cells and tissues, in order to be able to fully
exploit the high potential of cold plasmas for therapeutic
use.
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5. Acknowledgments
Prof. K.D. Weltmann (INP, Greifswald, Germany) and
Dr. F. Palumbo (CNR-IMIP) are acknowledged for
support and discussions. Mr S. Cosmai (CNR-IMIP) and
D. Benedetti (University of Bari) are acknowledged for
their technical contribution. The projects LIPP (Rete di
Laboratorio 51, Regione Puglia), RINOVATIS (PON
MIUR) are acknowledged for funding and supporting this
research.
6. References
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[5]G. Fridman et al. Plasma Processes Polym, 4, (2007).
[6]T. Von Woedtke et al. Physics Reports, (2013).
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[10]S.Kalaghatgi, et al. Annals of Biomedical
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[13]K.P. Arjunan et al. Plasma Processes Polym. 8,
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