Study of the effect on human mesenchymal and epithelial cells of an atmospheric
pressure plasma source driven by different voltage waveforms
F. Alviano1, L. Bonsi1,V. Colombo2,3, E. Ghedini2,3, M. Gherardi2, R. Laurita2, A. Liguori2,
C. Marchionni1, M. Marini1, F. Ricci 4, M. Rossi1, P. Sanibondi2, A. Stancampiano2, C. Zannini1
Alma Mater Studiorum-Università di Bologna
Department of Experimental, Diagnostic and Specialty Medicine
Via Belmeloro 8, 40126 Bologna, Italy
2
Department of Industrial Engineering (DIN)
3
Industrial Research Center for Advanced Mechanics and Materials (C.I.R.I.-M.A.M.)
Via Saragozza 8, 40123 Bologna, Italy
4
Immunohaematology and Transfusion Medicine Service, S.Orsola-Malpighi Hospital
Viale Aldo Moro 52, 40127 Bologna,Italy
1
Abstract: The aim of this work is the investigation of the effect of direct exposure to an
atmospheric pressure non-equilibrium plasma of human perinatal stem cells. A dielectric
barrier discharge (DBD) is used to generate a non-equilibrium plasma, driven by two different
high-voltage pulse generators. In order to compare the effect of the treatments, we
investigated cell survival and proliferation. Cells treated with plasma driven by nanosecond
high-voltage pulses showed a very high mortality rate even at the lower exposure time (1 s)
when the treatment was performed on cells deprived of the culture medium. Higher survival
and retention of proliferation were observed when cells were treated with the culture medium
or the culture medium alone was treated and then added to the cells. The effect of plasma
driven by microsecond high-voltage pulses was less cytotoxic when administered in
comparable conditions. A wound-healing assay was also performed. Preliminary results
suggest that treatment with microsecond high-voltage pulsed plasma enhances cell motility.
Keywords: Human perinatal stem cells, human mesenchymal cells, cell survival, cell
proliferation, atmospheric pressure non-equilibrium high-voltage pulsed plasma
1. Introduction
The aim of this work is the investigation of the effect of
direct or indirect exposure of human perinatal stem cells to
atmospheric
pressure
non-equilibrium
plasmas:
mesenchymal cells derived from fetal membranes
(FM-hMSCs) and epithelial stem cells derived from
amniotic membrane (hAECs). Preliminary experiments
performed with hAECs led to controversial results which
will not be shown here, leding us to focus this work on
FM-hMSCs. FM-hMSCs have a fibroblast like
morphology and easily proliferate in vitro; they express
typical mesenchymal markers and their surface marker
profile is comparable to that of bone marrow hMSCs.
Moreover, FM-hMSCs display some degree of
pluripotency as confirmed by the expression of some
specific stem cells markers and may be induced to
differentiate into different cell types.
Previous work, where a low power plasma, driven by a
micropulsed power supply, has been used for direct
treatment of cells [1], showed that non-thermal
atmospheric pressure plasma is not toxic to cultured
endothelial cells when administered for short times (up to
30 sec.); rather, cells were induced to synthesize FGF-2,
which stimulated their proliferation. The effect of plasma
treatment is likely ascribable to the peculiar ionic milieu
generated around the cells. It is well known that ROS/RNS
regulate a wide variety of signaling molecules that affect
most cell behavior, from proliferation to differentiation,
from cell arrest to apoptosis [2]. On the other hand, high
amounts of ROS/RNS may be very deleterious and induce
rapid cell death. Given the interesting properties of
FM-hMSCs, we started to investigate different modes of
plasma treatment in order to examine how plasma
exposure might affect FM-hMSC proliferation and
differentiation. The present work is preliminary to more
in-deep studies that will try to correlate the composition of
the ionized gas delivered by plasma treatment and the cell
responses at the molecular level.
2. Materials and Methods
2.1 Plasma sources
A dielectric barrier discharge is used to generate a
non-equilibrium plasma, driven by two different
high-voltage pulse generators: the first one having peak
voltage (PV) between 15 and 25 kV, repetition rate (RR)
between 50Hz and 3500 Hz, pulse duration between 1µs
and 10 µs and rise time of about 5 µs, while the other
generates a voltage with a PV between 7 and 20 kV, a RR
between 50 and 1000 Hz, a pulse duration about 40 ns and
rise time 3 ns.
2.2 Cell preparation
FM-hMSCs were derived from term placentas of healthy
donor mothers undergoing caesarean sections as
previously described [3, 4]. All tissue samples were
obtained after informed written consent. To analyze the
effect of non-equilibrium plasma, the isolated cells were
seeded in 24-well plates at a concentration of 13.000
cells/cm2. Cells were maintained in Dulbecco's Modified
Eagle Medium (DMEM) supplemented with 10% Fetal
Bovine Serum (FBS) and 1% penicillin-streptomycin and
incubated at 37° C and 5% CO2 to allow adherence and
expansion.
2.3 Cell culture treatments
Cells were seeded in 24-well plates 24 hrs before plasma
treatment. Three modalities of plasma treatment were
compared: i) the culture medium was removed and the
cells were treated with plasma. The culture medium was
then returned to the wells; ii) cells were treated with
plasma without previous removal of medium (0.4 mL); iii)
the culture medium (0.4 mL) was removed, treated with
plasma, then returned to the wells. The gap between the
source tip and the cells was 2 mm for the direct treatment
of cells, while 1 mm gap was implemented for all the other
cases. In initial experiments with direct exposure of cells to
the plasma exposure time ranged from 1 to 20 seconds,
then 1-second treatment was chosen. Cells were cultured
and monitored for the following 7 days in a medium
supplemented with 10% Alamar Blue.
2.4 Alamar Blue assay for evaluation of cell viability
and proliferation
The Alamar Blue assay is designed to quantitatively
measure the proliferation of various human and animal cell
lines. Alamar Blue is an indicator dye which incorporates
an oxidation-reduction indicator that both fluoresces and
changes colour in response to chemical reduction of
growth medium, resulting from cell growth. At prefixed
times, cell plate fluorescence is read in a Victor-2™ plate
reader at a fluorescence excitation wavelength of 570 nm
and absorbance wavelength 590 nm.
2.5 Trypan blue cell viability assay
In vitro assessment of cell viability and proliferation
was perfomed by Trypan Blue exclusion assay [5]. In this
assay, cells are detached from plate and counted in an
hemocytometer chamber, where necrotic cells appear blue
stained.
2.6 Flow cytometer evaluation of apoptosis
Viability was evaluated with Annexin V Apoptosis
Detection Kit FITC according to the instructions of
Immunological Sciences and the staining was analyzed on
Beckman Coulter's Navios FC. Early apoptotic cells were
defined as PI-negative and Annexin V-positive.
Annexin-V and PI-positive cells were either late apoptotic
or necrotic.
2.7 Wound Healing Assay
The wound-healing assay is based on the creation of a
scratch (“wound”) on the cell monolayer. FM-hMSCs
used for the assay were seeded at a concentration of
25.000 cells/cm2 and maintained in DMEM 10% FBS.
After the scratch creation, the cells were treated with
microsecond pulsed plasma, with or without medium, and
treated and untreated cultures were monitored at regular
intervals for their capacity to “close the wound”.
Microsecond pulsed plasma was delivered according to
two different frequency/voltage combination: 500 Hz and
17 kV or 500 Hz and 20 kV, 30 s exposure time. As in the
cell treatment, 2 mm gap between the source and the cells
was adopted for exposure without any medium, while 1
mm between the source and the solution was used in the
other test. Migration was evaluated by using the Image
Analyzer Software ImageJ®, which measures the scratch
area.
Migrating and proliferating cells progressively occupy
the scratch area; the migration rate is expressed as the
ratio of newly-colonized area to time.
3. Results
3.1 Nanosecond pulsed Dielectric Barrier Discharge
Cells were treated for 1 to 20 seconds with nanosecond
pulsed plasma (25,4 kV and 500 Hz) without interposed
culture medium. Some residual viability and proliferation
was displayed only at the lower exposure time (Fig.1).
Fig.1 Effect of direct exposure to nanosecond pulsed plasma on
FM cell viability assessed by Alamar Blue assay following 7
culture days.
Subsequent investigations were performed only at 1
second exposure time. As shown in fig. 2, about 3.5% of
cells were viable immediately after the treatment (T0),
plasma treatment, viable cells were further reduced, as the
percentage of dead cells was respectively 40 and 45% of
residual cells compared to untreated cells.
Fig.2. Effect of 1 second-direct exposure to nanosecond
pulsed plasma on FM cell viability and proliferation evaluated
by Trypan Blue assay
Cells exposed to nanosecond pulsed plasma with the
interposition of culture medium displayed no cytotoxic
effect at T0. Cells did not proliferate for the following 24
hrs. Seventy-two hours after plasma treatment a very
small cell growth could be observed, with 40% of
apoptotic and necrotic cells (Fig. 3).
When culture medium alone was treated with
nanopulsed plasma, cells displayed at 24 hours a
relatively higher percentage of apoptotic cells compared
to untreated sample, respectively 54 vs. 12%. Six days
from the treatment 98% of cells were apoptotic or
necrotic (Fig. 3).
Fig.3. Effect of exposure of cells with culture medium
interposition and of exposure of culture medium alone to
nanosecond pulsed plasma on FM cell viability and proliferation.
Data are presented as a combination of flow cytometer analysis
and Trypan Blue assay
3.2 Microsecond pulsed Dielectric Barrier Discharge
The effect of a microsecond pulsed plasma (PV of 20 kV
and PRR of 1000 Hz) was investigated using the same
modalities of cell or medium exposure used with
nanosecond pulsed plasma. Only the 1 second exposure
time was used. Following direct exposure to microsecond
pulsed plasma, about 50% of cells were viable, compared
to untreated cells (Fig.4)
In general, the effect of microsecond pulsed plasma was
significantly less pronounced than that of nanosecond
pulsed plasma. In particular, cells were not hampered in
their growth rate, which was similar to that of untreated
cells, at least for the first 72 hrs. (Fig.4).
Fig.4. Effect of direct exposure to plasma microsecond pulsed
power supply on FM cell viability and proliferation evaluated by
Trypan Blue assay
At subsequent times, cells stopped growing even though
they did not reach a confluent state.
Cells exposed to plasma microsecond-pulsed power
supply with the interposition of culture medium displayed
no apparent cytotoxic effect at T0. At following time
points, cell growth rate was slower than that of untreated
controls, with similar percentage of apoptotic cells. Cell
viability and proliferation rate following the exposure of
culture medium to micropulsed plasma did not
significantly differ from that of untreated sample.
Fig.5. Effect of exposure with culture medium interposition and
of culture medium exposure to plasma microsecond pulsed power
supply on FM cell viability and proliferation evaluated by flow
cytometer analysis
3.3 Wound healing assay
The wound healing assay was carried out as described
above. No cell migration was observed with 500 Hz and 20
kV treatment in the absence of culture medium, owing to
the cytotoxic effect developed. The other treatment
conditions were compatible with cell migration and
proliferation. Treatment of FM-hMSCs in the presence of
culture medium with the plasma device set to 500 Hz and
17 kV apparently increased the migration rate about 1.65
fold compared to control untreated cells. Data are shown in
Fig. 6, while Fig. 7 shows a image of the plate. Further
experiments are needed in order to assess whether
differences in migration rate are statistically significant.
Ctrl
A
A direct
B
B direct
Fig.6. Effect of exposure with culture medium interposition
(A,B) and of direct exposure (A direct, B direct) to different
plasma microsecond pulsed power supply conditions on FM cell
migration rate evaluated by the wound healing assay. A=500 Hz
and 17 kV, B= 500 Hz and 20 kV
Fig.7. Representative image of in vitro wound healing of
FM-MSCs
4. Conclusions
Though preliminary, these data demonstrate that
FM-hMSCs are differently affected by nanosecond pulsed
or microsecond pulsed plasma. In fact, when treated with
nanosecond pulsed plasma, deleterious effects
predominate, although treatment of the cells in the
presence of culture medium allowed for a very limited
recovery of the proliferative capacity. As shown in Fig. 8,
most viable (i.e. not necrotic) FM-hMSCs appear either
apoptotic (“blebbing”surface) or senescent (enlarged size).
Fig. 8. Representative image of hemocytometer chamber count.
(h:healty cells; s: senescent cells; a: apoptotic cells; n: necrotic
cells)
Senescence is a condition where cells, though viable, are
unable to proliferate, owing to severe DNA damage;
senescent cells may damage neighboring cells by secreting
pro-inflammatory cytokines [6]. We are planning further
experiments to better characterize the effects of
nanosecond pulsed plasma to FM-hMSCs, it is however
obvious that it has both cytotoxic and cytostatic effects in
all the tested conditions. Several studies [7-9] highlighted
that the cell exposure to a nanosecond pulsed electric field
can interact with subcellular structures, inducing apoptosis
and physical damage to the DNA, while a microsecond
pulsed electric field, mostly interacts with the cell
membrane.
On the other hand, microsecond pulsed plasma, at 1 sec.
exposure time, has limited or null cytotoxic effects in all
the tested treatment conditions. At variance with the
growth-promoting effects exerted by non-thermal
atmospheric pressure DBD plasma on endothelial cell
culture [1], a delayed cytostatic effect could be observed in
FM-hMSCs in all the tested treatment conditions (Figs. 4
and 5). This intriguing result warrants further
investigations, since it might be indicative of either a
delayed attainment of senescence or, most interestingly, of
the exit from the stemness state concomitant with the entry
into a differentiation pathway. The latter would involve the
modulation of specific gene expression.
Finally, the wound healing assay, carried out with a DBD
plasma device under particular operating conditions,
suggests that FM-hMSCs mobility might be modulated by
the ionic discharge generated by microsecond pulsed
plasma. Should this very preliminary result be confirmed
by further experiments, it deserves great attention, since
such short-term responses are generally the result of
post-translation protein modifications, i.e. the involvement
of biomolecular mechanisms different from those
hypothesized to occur as a consequence of the
microsecond pulsed plasma exposure in non-confluent
cultures (please see the paragraph above).
In any case, the effects of atmospheric pressure plasma on
living cells should be studied within a rigorous
characterization of the ionic environment produced and of
the reactive/radical species involved. Future studies by our
group will address this issue as well as those outlined
above.
5. References
[1] S.U. Kalghatgi et al., 31st Annual International
Conference of the IEEE EMBS Minneapolis, Minnesota,
USA, September 2-6, 2009.
[2] V.J. Thannickal et al., Am J Physiol Lung Cell Mol
Physiol 279, L1005 - L1028, 2000.
[3] C. Ventura et al., J Biol. Chem., 282 (19), 14243-52,
2007.
[4] O. Parolini et al., Stem Cells, 26(2), 300-311, 2008.
[5] S. A. Altman et al., Biotechnol. Prog., 9 (6), 671-674,
1993.
[6] J. Campisi et al., Cell, 120 (4), 513-522, 2005.
[7] J. Deng et al., Biophysical Journal, 84 (4), 2709–2714,
2003.
[8] K. H. Schoenbach, IEEE Transactions on Dielectrics
and Electrical Insulation 14, 5, 2007.
[9] M. Stacey et al., Mutation Research 542, 65-75, 2003.