The role of liquid environment as modulating medium for plasma-cell
interactions
Thomas von Woedtke1, Susanne Blackert2, Beate Haertel2, Manuela Harms2, Ulrike Lindequist2,
Katrin Oehmigen1, Kristian Wende1,3, Klaus-Dieter Weltmann1
1
Leibniz Institute for Plasma Science and Technology (INP Greifswald),
2
Institute of Pharmacy of the Ernst Moritz Arndt University Greifswald,
3
Centre for Innovation Competence plasmatis, Greifswald, Germany
Abstract: From experiments on inactivation of bacteria in liquids using surfaceDBD plasma it is known that bactericidal plasma effects depend strongly on
changes of the liquid environment like pH decrease as well as formation of
oxygen and nitrogen-containing chemical species. In the study presented here it
is demonstrated that the influence of plasma treatment on cellular characteristics
like cytotoxicity, cell attachment, or intracellular occurrence of reactive oxygen
species (ROS) depends not only on characteristics of the liquid environment
where the cells are suspended. Moreover, changes of the liquid environment
itself make significant contributions to plasma-induced biological effects.
Keywords: plasma medicine, dielectric barrier discharge, plasma-liquid
interactions, plasma pharmacology
1. Introduction
Under physiological conditions as well as during invitro culture, cells are surrounded by an aqueous
liquid compartment which is essential for cell
survival. Therefore, if cells are treated by
atmospheric pressure plasma, direct plasma effects
on cells cannot be separated readily from indirect
effects caused by changes of the liquid environment.
It was demonstrated recently that inactivation of
microorganisms suspended in aqueous liquids by
surface dielectric barrier discharge (surface-DBD)
plasma is supported by acidic conditions and it was
assumed that bactericidal activity of nitrogen and
oxygen-based reactive species which are generated
in the liquid is modulated by low pH [1-3].
The aim of this paper is to get more insight into the
role of the liquid environment of living cells for
biological effects induced by atmospheric pressure
plasma treatment.
2. Experimental
The surface-DBD arrangement schematically
shown in Fig. 1 was based on a setup described
elsewhere [1].
Figure 1. Schematic drawing of the surface DBD arrangement
All experiments are performed at ambient air
conditions using a pulsed sinusoidal voltage of
10 kVpeak (20 kHz) with a 0.413/1.223 s plasmaon/plasma-off time. Energy of 2.4 mJ was
dissipated into the plasma in each cycle of high
voltage. The power was 0.25 W ⋅ cm-2. There is no
direct plasma-liquid contact.
Detection of intracellular reactive oxygen species
(ROS) was realized immediately after plasma
treatment of suspended cells by fluorescence assay.
In presence of ROS the fluorescent 2‘,7‘-dichloro
fluoresceine (DCF) is formed 2‘,7‘-dichlorodihydro-
fluoresceine diacetate (H2DCF-DA) which is able to
pass through the membrane of living cells. Cells in
suspension were incubated with 10µM H2DCF-DA
for 30 min immediately after plasma treatment. After
extensive washing, cells were finally pelleted by
centrifugation, resuspended in HBSS and 200 µl
each inoculated into a well of black 96-well plate
(nunc). Fluorescence intensity was measured at
Ex485nm/Em525nm for 90 min. Intracellular ROS
abundance was calculated as percent of control.
Escherichia coli NTCC 10538 are plasma treated
in sodium chloride solution (NaCl 0.85 %). Either,
5 ml of NaCl solution were plasma treated and
immediately (t < 10 s) afterwards added to the
microorganisms and allowed 15 min to soak. Or
E. coli suspended in NaCl solution were plasma
treated directly. Number of viable microorganisms
(cfu ⋅ ml-1) was estimated by the surface spread
plate count method using aliquots of serial
dilutions of microorganism suspensions in NaCl
solution
according
to
the
European
Pharmacopoeia.
3. Results and discussion
Einfluss der DBE-Behandlung auf HaCaTs in RPMI
140
120
100
Vitality
Vitalität [%]
[%]
Cells of he nontumorigenic human keratinocyte cell
line HaCaT [4] were grown in (a) RPMI 1640
nutrient medium with L-glutamine supplemented
with 8% FCS and 1% penicillin-streptomycin
solution (10,000 IU/ml penicillin; 10,000 µg/ml
streptomycin), or (b) in IMDM medium with Lglutamine and 8% FCS and antibiotics (see a). Cells
were maintained at 37°C in a humidified atmosphere
of 5% CO2 and 95% air and were sub-cultured
routinely. For direct treatment of suspended cells
with DBD plasma, 106 cells were plated on 60 mm
diameter Petri dishes in 4 ml nutrient medium. After
plasma treatment, cells were either kept with the
treated medium or got fresh medium. For indirect
treatment, 4 ml nutrient medium without cells were
treated with DBD and thereafter the cells were
immediately plated into the treated medium. Cells
were cultured at 37°C in a humidified atmosphere of
5% CO2 and 95% air to allow cell attachment on the
bottom of the cell culture plates. After removing the
medium adhered cells can be detached by
subsequent treatment with PBS/EDTA (10 min) and
trypsin/EDTA in Ca2+/Mg2+-free PBS (final
concentration: 0.05%/0.1%; 5 min) at 37°C,
centrifuged, and the cell pellet can be resuspended in
PBS supplemented with 0.1% NaN3 and 1% FCS.
Number of attached cells was calculated using a
Neubauer chamber. Cytotoxicity was estimated by
neutral red uptake (NRU) assay which is based on
the trapping of the dye into (acidic) lysosomes of
vital cells [5]. 50µl of the either in RPMI or IMDM
treated cell suspension was inoculated into a well of
a 96 well plate and left undisturbed in an incubator
for 48h, 72h, or 96h. After incubation, cell culture
medium was replaced with neutral red media (33µg/
ml) and incubated for 3 hours at 37°C. Cells were
washed with Hanks buffered salt solution (HBSS)
and lysed using acidified ethanol (1% acetic acid in
50% ethanol/water). Optical density was measured
at 550 nm and vitality was calculated as percent of
vehicle control.
80
60
40
20
0
Kontrolle
control
10sec
20sec
48h
30sec
72h
50sec
100sec
96h
Figure 2. Vitality of HaCaT keratinocytes (% relative to
untreated control) 48, 72 and 96 h after 10-100 s surface-DBD
plasma treatment of cell suspensions in RPMI nutrient medium;
estimation of vitality by neutral red uptake (NRU) assay.
Fig. 2 shows the percentage of vital HaCaT
keratinocytes suspended in RPMI 48-96 h after
surface-DBD plasma treatment in atmospheric air
using different treatment times. Whereas 10 and 20 s
plasma treatment resulted in even a slight increase of
the number vital cells compared to untreated control,
30, 50 and 100 s plasma treatment caused
progressive reduction of number of vital cells.
Einfluss der DBE-Behandlung auf HaCaTs in IMDM
140
Vitality
[%]
Vitalität [%]
120
100
80
60
40
20
0
control
Kontrolle
10sec
20sec
48h
30sec
72h
50sec
100sec
96h
Figure 3. Vitality of HaCaT keratinocytes (% relative to
untreated control) 48, 72 and 96 h after 10-100 s surface-DBD
plasma treatment of cell suspensions in IMDM nutrient medium;
estimation of vitality by neutral red uptake (NRU) assay
However, if the same experiment is realized using
HaCaT keratinocytes suspended in IMDM, even
100 s surface-DBD plasma treatment did not induce
any changes of cell vitality (Fig. 3).
Intensity of fluorescence
% von Kontrolle
[% related to control]
Differences dependent on the nutrient medium in
which cells were suspended during plasma treatment
have been found for the intracellular occurrence of
reactive oxygen species (ROS), too (Fig. 4).
analytical error, increase of intracellular ROS
content was detected in cells which were suspended
in RPMI, only. In contrast to that, cells suspended in
IMDM did not show any change in intracellular
ROS concentration. Consequently, dependent on
special characteristics of the liquid environment,
cells are protected from detrimental plasma effects
or not.
Similar effects have been found with influences of
surface-DBD treatment on intracellular DNA (data
not shown; see S. Reuter et al., this conference).
One reason for cell protection from plasma-caused
cytotoxicity is a higher antioxidative capacity of
IMDM compared to RPMI which was confirmed by
a special test method called Trolox equivalent
antioxidative capacity (ORAC, oxygen radical
absorbance capacity) assay (data not shown).
However, liquid environment of cells has not only
protective or more generally, modulating effects if
cells in suspension are directly treated by surfaceDBD plasma. Moreover, plasma treatment of liquids
and subsequent addition to cells can also cause
significant biological effects.
300
IMDM
250
direct + ME
RPMI
direct
Indirect
200
150
100
50
0
30
50
80
100
120
-50
DBE in Medium
für ...stime [s]
Plasma
treatment
Figure 4. Relative intracellular ROS content of HaCaT
keratinocytes immediately after treatment of cell suspensions in
IMDM or RPMI nutrient medium, respectively, by surface-DBD
in atmospheric air
Intracellular
ROS
content
was
measured
immediately after plasma treatment of cells
suspended in IMDM or RPMI, respectively, by
surface-DBD. Apart from the results after 50 s
plasma treatment which are probably caused by an
Figure 5. Number of adhered HaCaT keratinocytes 24 h after
treatment of cells suspended in RPMI nutrient medium by
surface-DBD plasma; plasma treatment time 20-120 s; direct
(red line) = plasma treatment of cells suspended in nutrient
medium; Indirect (blue line) = Plasma treatment of nutrient
medium, subsequent addition of cells; direct + ME (green line) =
plasma treatment of cells suspended in nutrient medium,
medium exchange 15 min after plasma treatment
To demonstrate this, cells suspended in RPMI were
treated directly by surface-DBD plasma. In a second
test series, RPMI nutrient medium without cells was
plasma treated but immediately afterwards cells
were plated into the medium. In a third test series,
cells suspended in RPMI were treated directly by
surface-DBD plasma, too, but 15 min after treatment
nutrient medium was replaced by fresh non-treated
4. Conclusions
Such transmission of plasma effects on cells via
liquid media was demonstrated in another
experiment using suspended microorganisms
(Fig.6).
References
Number of viable microorganisms [cfu . ml-1]
medium (ME, medium exchange). In all cases,
number of attached (living) cells was detected 24 h
after plasma treatment. Results are shown in Fig. 5.
Obviously, the recovery of cells was significantly
lower after 20 s plasma treatment already, and was
further decreased by increasing plasma treatment
time. But, surprisingly, the same effect was induced
if the nutrient medium was plasma treated and the
cells were added subsequently. Consequently, at
least in this case the dominating cause of plasmainduced biological effects seems to be not a “direct”
plasma-cell interaction but a change of the liquid
environment inducing secondary effects on cells.
This was underlined by the fact that an exchange of
nutrient medium 15 min after plasma treatment
resulted in a higher recovery of cells compared to
directly or indirectly treated cells.
9
1,00E+08
8
.
1,00E+09
10
10
7
1,00E+06
6
10
1,00E+05
10 5
1,00E+04
10
4
1,00E+03
10 3
1,00E+02
2
1,00E+01
1
10
10
1,00E+00
10
detection limit
0
0
2
4
6
8
10
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Wilke, K.-D. Weltmann, Th. von Woedtke, Plasma
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plasma treated NaCl solution added to E. coli
plasma treated E. coli suspension
plasma treated E. coli suspension
1,00E+07
10
Results presented here underline that the vital
liquid environment of cells is playing a
dominating role in the transmission of biological
effects from atmospheric pressure plasma to living
cells or microorganisms, respectively. On the one
hand, special characteristics of these liquids, e.g.
its antioxidative capacity can protect cells from
detrimental plasma effects. On the other hand,
plasma-induced changes of the liquid itself can be
identified to be a dominating cause of plasma
activity on living systems. Above all the latter
aspect will open the door to the new field of
plasma pharmacology, i.e. the plasma-supported
generation and/or optimization of active substance
containing liquids for medical applications.
12
Plasma treatment time [min]
Figure 6. Inactivation kinetics of E. coli as a result of direct
plasma treatment of bacteria-containing sodium chloride (NaCl)
solution (green dotted line; ) or of 15 min impact of plasmatreated NaCl solution added immediately after plasma treatment
over different periods of time (red line; )
Surface-DBD treatment of E. coli suspended in
non-buffered NaCl solution resulted in a complete
bacteria inactivation (≥ 7 log) within 5 min. But, if
a 7 min plasma-treated NaCl solution was added
immediately after plasma treatment to E. coli and
allowed to soak for 15 min, nearly the same
complete bacteria inactivation was found [2].
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Akishev (eds.), Plasma for Bio-Decontamination,
Medicine and Food Security, Nato Science for Peace
and Security Series A, Springer, submittted, under
review
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Acknowledgement: This work was supported within
the joint research project “Campus PlasmaMed” by
the German Federal Ministry of Education and
Research.