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Dose-dependent killing of leukemia cells by low-temperature plasma

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2012 J. Phys. D: Appl. Phys. 45 422002
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IOP PUBLISHING

JOURNAL OF PHYSICS D: APPLIED PHYSICS

J. Phys. D: Appl. Phys. 45 (2012) 422002 (6pp)

doi:10.1088/0022-3727/45/42/422002

FAST TRACK COMMUNICATION

Dose-dependent killing of leukemia cells

by low-temperature plasma
N Barekzi and M Laroussi
Laser and Plasma Engineering Institute, Old Dominion University, Norfolk, VA 23529, USA
E-mail: nbarekzi@odu.edu and mlarouss@odu.edu

Received 13 July 2012, in final form 31 August 2012

Published 4 October 2012
Online at stacks.iop.org/JPhysD/45/422002
Abstract
The effect of low-temperature atmospheric pressure plasma towards the progression of
cancerous human T-cell leukemia cells was investigated. The plasma pencil, which utilizes
short duration high voltage pulses, was used to generate a low-temperature plasma (LTP)
plume in ambient air. Our data showed that cell morphology and cell viability were affected in
a dose-dependent manner after treatment with LTP. The outcome of this study revealed that the
effect of plasma exposure was not immediate, but had a delayed effect and increasing the time
of plasma exposure resulted in increased leukemia cell death.
(Some figures may appear in colour only in the online journal)

patients and their families, we propose to use a device termed

the plasma pencil to generate non-thermal gas plasma, also
known as low-temperature atmospheric pressure plasma or
low-temperature plasma (LTP) for treatment of cancerous
leukemia cells.
The plasma pencil is a device that produces an LTP plume
or jet using inert gas in ambient pressure [15]. The LTP that
is generated can be used at biologically tolerant temperature
regimes of 2540 C as a tool for the treatment of heatsensitive materials such as mammalian cells and biological
samples. The plasma plume can be touched, is electrically
safe and does not cause any heating or painful sensation to
the skin or gums [15]. The plasmas that are generated by the
plasma pencil are sources of reactive oxygen species (ROS)
and reactive nitrogen species (RNS). These include hydrogen
peroxide (H2 O2 ), and other short-lived and long-lived reactive
species, such as O, O2 , O
2 , O3 , OH, NO and NO2 which
were previously studied and determined in our laboratory by
various diagnostic techniques including emission spectroscopy
analysis [1521]. The properties of LTP such as the low
temperature, ambient pressure and reactive species generated
by the plasma pencil have been positively exploited in our
previous studies involving recombinant -Synuclein amyloid
fibrils [16], eukaryotic microalgae [22], prokaryotic cells [13]
and dental applications [8]. In this paper, the effect of LTP

1. Introduction
The use of non-thermal gas plasmas in medicine has become
an increasingly important topic of research for physicists,
biologists and medical personnel alike. Many devices that
generate non-thermal gas plasmas have been shown to be
efficacious in a variety of studies that include bacterial
inactivation [13], tissue and surface sterilization [4], blood
coagulation [5], dental treatments [68] and skin wound
healing [9, 10]. In addition, preliminary investigations into
the use of non-thermal gas plasmas as a therapeutic treatment
against various cancer cell lines have yielded promising results
[11, 12]. In this paper, we further explore the prospect of using
non-thermal gas plasma as a possible alternative treatment
against the blood disease leukemia for the first time.
Leukemia is the most common childhood malignancy and
accounts for approximately 1 out of 3 cancers in children
[13]. Even though leukemia in certain cases is curable
through chemotherapy, radiation therapy or bone marrow
transplantation, some drug therapies may increase the short
term patient survivability, but still have high rates of long
term morbidity [14]. The prognosis of leukemia depends
on numerous factors and there is no way to prevent the
disease. As an alternative to traditional therapies that could
result in detrimental side-effects and increased burden to the
0022-3727/12/422002+06$33.00

2012 IOP Publishing Ltd

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J. Phys. D: Appl. Phys. 45 (2012) 422002

Fast Track Communication

Figure 1. (a) Schematic and (b) photograph showing the plasma pencil and the application of low-temperature atmospheric pressure plasma
in the in vitro treatment of leukemia cancer cells in a tissue culture plate.

in the kilohertz range. In order to enhance the plasma

chemistry while maintaining a low gas temperature, very short
(nanoseconds to microseconds) voltage pulses can be applied
at a reduced duty cycle. Using the DBD concept in a cylindrical
geometry and injecting a gas such as helium at a certain flow
rate it is possible to launch a plasma plume outside the confines
of the electrodes and into ambient air. This is the concept upon
which the design of the plasma pencil is based [15]. Although
the gas flow is important, the plasma plume was found to be
electrically driven and in fact the plume consists of a train of
high-speed plasma packets known as plasma bullets [20, 21].
The plasma pencil is made of a 25 mm diameter hollow
dielectric tube with two copper ring electrodes each attached
to the surface of a dielectric alumina disc. These discs
are separated by a gap distance of 5 mm and the centrally
perforated diameter of the hole is 3 mm. The copper ring
electrodes have a smaller outer diameter than the alumina
discs and a larger perforation than the 3 mm hole. The copper
ring electrodes are connected to a high-voltage (HV) pulse
generator. Figure 1 shows a schematic (a) and photograph (b)
of the plasma pencil operating at atmospheric pressure. The
CCRF-CEM leukemia cells are suspended in media solution
and treated with LTP. The plasma pencil is oriented above
each well of the cell culture plate which is placed on an
adjustable platform. The distance between the nozzle of the
plasma pencil and the top of the media was kept constant at
15 mm. Conditions for the plasma pencil shown in figure 1
can be changed in order to obtain a characteristic plume with
unique enhanced gas-phase chemistry. For this study, the

treatment on a single tissue culture cell line of leukemia cells

has been investigated to determine the antitumour activity of
LTP and the long term effects of plasma induced cell death.

2. Experimental section
2.1. Cell line and cell culture
The human T-cell line (ATCC Number CCL-119; aka CCRFCEM) isolated from the peripheral blood of a 4 year old
female child (CEM) with acute lymphoblastic leukemia was
maintained according to standard protocols. Briefly, the
CCRF-CEM cells which are non-adherent leukemia cancer
cells were grown in complete growth media in 75 cm2 vented
sterile polystyrene tissue culture flasks at 37 C in a humidified
atmosphere containing 5% CO2 . The complete growth
media consisted of RPMI-1640 media supplemented with 10%
fetal bovine serum, 1% antibiotics (Penicillin/Streptomycin)
and 1% glutamine. RPMI-1640 media utilizes a sodium
bicarbonate buffering system to maintain neutral pH between
7.0 and 7.4.
2.2. Plasma treatment of CCRF-CEM cells
The plasma pencil generates a plume or jet of non-equilibrium
plasma at atmospheric pressure and low biologically tolerant
temperature using the dielectric barrier discharge (DBD)
concept. Traditional DBDs employ two electrodes insulated
by a dielectric material. The DBD plasma formation ensues as
a result of applied voltage in the kilovolts range at frequencies
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J. Phys. D: Appl. Phys. 45 (2012) 422002

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Figure 3. A photograph of the trypan blue exclusion assay showing

the live versus dead cells after 3 min of exposure to plasma treatment
sampled at 12 h post-treatment. A ratio of 1 : 1 of the trypan blue dye
(0.4% concentration) to 106 cells in media was used and the mixture
was loaded on a hemocytometer. The cells that are dead absorb the
trypan blue dye while live cells do not. The difference is clearly
observed using a bright field microscope at a magnification of 25.

Figure 2. A photograph of CCRF-CEM leukemia cancer cells

growing in complete RPMI growth media inside of a 75 cm2 vented
sterile polystyrene tissue culture flasks at 37 C in a humidified
atmosphere containing 5% CO2 . The picture was taken using an
inverted bright field microscope at a magnification of 10.

following parameters were used to generate LTP using the

plasma pencil: He as the feed gas at a flow rate of 5 l min1 or
slm; HV was set at 7.5 kV; frequency set at 5 kHz and the pulse
width was set at 0.50 s (500 ns). Plasmas generated under
these conditions have been fully characterized and published
previously including the identification of the various reactive
species that are generated [1521].
Prior to plasma treatment, the CCRF-CEM cells were
grown for three days to reach a cell concentration of 2 106
cells per ml. Figure 2 is a photograph showing the CCRFCEM cells growing in suspension of complete growth media
inside of a 75 cm2 vented cell culture flask. The characteristic
morphology of the CCRF-CEM cells is immature small to
intermediate round or oblong cells. The cells are precursor
lymphocytes which are clearly non-adherent white blood cells
in solution with 12 nucleoli in the nucleus and a very small
cytoplasm. This figure demonstrates that the cells are freely
floating in the media and that the LTP reactive species can
interact with the cells to influence the viability of the cells.
At the appropriate cell density, the cells were harvested by
centrifugation at 1000 rpm for 10 min at room temperature.
The cells were washed with sterile phosphate-buffered saline
(0.2M phosphate, 1.5M NaCl, pH 7.4) and re-suspended
in complete growth media. A total of 1 106 cells ml1
was seeded onto each well of a 24-well cell culture plate.
Subsequently, each well containing the cells in complete RPMI
growth media was oriented under the plasma pencil on the
adjustable platform and exposed to different doses of LTP
as measured by treatment time. Helium gas at a flow rate
of 5 slm was used as the working gas. The control samples
were treated with only the non-ionized helium gas alone. In
both the controls and LTP treated samples, the pH of the
media were measured using pH strips (EM Science) and an
Orion pH meter (Thermo scientific). The pH remained neutral
supporting the hypothesis that the effects of the ROS and RNS
on the cells are not via pH change. The experiments were
carried out in triplicate at room temperature. After plasma

treatment, the culture plates containing the cells were returned

to the incubator at 37 C containing 5% CO2 . The cells were
sampled at 0 h, 12 h, 36 h and 60 h post-plasma treatment for
cell viability to determine the antitumour activity of a single
dose of LTP.
2.3. Cell viability assay
The viability of the CCRF-CEM cells was determined using
trypan blue exclusion assay to determine the cellular death
and viability. A ratio of 1 : 1 of the trypan blue dye (0.4%
trypan blue solution from Amresco) to cells in media was
used. The trypan blue dye cannot penetrate the intact cell
membrane of live cells. However, cells that are dead absorb
the trypan blue and can be observed as a distinct blue coloured
cell under the microscope (figure 3). The results in figure 3
reveal that the LTP reactive species have disrupted some of
the cell membranes. The cell morphology reveals no longer
intact, but broken membranes, thus allowing the entry of the
trypan blue dye. In order to quantitatively show the effect of
LTP on the cells, the trypan blue-cell mixture was loaded onto a
hemocytometer and both live and dead cells were counted using
a phase-contrast bright field microscope. The total numbers
of dead and live cells were enumerated and the percentages
of total viable cells were calculated. Since cells dye naturally
in tissue culture, the percentage of dead cells in the control
non-plasma helium-only treated samples were subtracted for
every LTP exposure time point. The non-plasma-treated cell
viability counts provided the background cell counts that were
used to normalize the total cell viability and reflect more clearly
the effect of LTP on the cells. The normalized overall cell
viability was plotted as mean percent survival over time. Cell
death and viability were monitored over a 2.5 day period. The
dose response and long-term cell viability as a function of
treatment exposure time is shown in figure 4. The CCRF-CEM
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J. Phys. D: Appl. Phys. 45 (2012) 422002

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the cells were evaluated for cell survival after the single dose
of LTP treatment at 0, 12, 36 and 60 h post-plasma treatment.
The experimental setup allowed for determining the single dose
response and long-term killing effect of LTP against cancerous
leukemia cells.
2.4. Data and statistical analysis
The Microsoft Excel 2010 data analysis software was used
for statistical analysis. Unless otherwise noted, the data are
expressed as the arithmetic mean standard deviation of
three separate experiments. Viability results were expressed as
median bacterial count reduction in percent and the statistical
significance of the results was calculated using the Students
t test. Differences were considered statistically significant at
p values <0.05 and highly statistically significant at p values
<0.001.

3. Results and discussion

The applications of low-temperature atmospheric pressure
plasmas (LTP) are alluring in the biomedical arena because of
the biologically tolerant temperature regimes. In addition, the
unique characteristic of non-thermal gas plasmas enhanced
gas-phase chemistry can be positively exploited. The reactive
chemical species are obtained when the electrons undergo
collisions with the background gas and facilitate the formation
of radical and metastable states. As a result, the plasma does
not cause any thermal damage, since the heavy species such
as the ions and neutrals remain within the biologically tolerant
temperature regime [23]. In this study, helium was used as
the working gas. In this scenario, collisions between the
energetic electrons and the background gas produce excited
and metastable helium atoms. These metastable helium atoms
interact with the nitrogen and oxygen molecules that diffuse
into the plasma plume from the surrounding air to produce
different oxygen and nitrogen based reactive species. In
general, the lifetime of the charged particles is very short
and can be shorter or comparable to the applied pulse width.
The short-lived and long-lived reactive species include radicals
such as O, O
2 , OH, NO and NO2 and non-radicals such as
H2 O2 and O3 [15] which have an effect on the leukemia cells
presented in this study.
The leukemia cells are non-adherent and typically found
suspended in solution. This unique characteristic of the cancer
cells make them ideal targets for investigating LTP treatment
without removal of the media. Treating cells in complete
growth solution mimics the host environment more closely
than cells treated in the absence of media. Furthermore, the
dose of LTP is an important parameter to induce cell death.
Therefore, our first experimental setup involved determining
a plasma dose response killing curve using the CCRF-CEM
leukemia cells in vitro. The results from the cell viability assay
are shown in figures 4(a)(c). The three data sets show the
results of cell viability counts using the trypan blue exclusion
assay. Cells that are alive have an intact cell membrane and
therefore do not let the trypan blue dye enter. However,
cells that have been treated with LTP usually have a disrupted

Figure 4. The effect of varying exposure time of plasma treatment

on the viability of CCRF-CEM cells. The cell viability was
determined using trypan blue exclusion assays and the values are
expressed as the mean percentage of total viable cells standard
deviation of three separate experiments. (a) cell viability
determined at 12 h post-plasma treatment, (b) cell viability
determined at 36 h post-plasma treatment, (c) cell viability
determined at 60 h post-plasma treatment.

leukemia cells were exposed to a single dose of plasma for 0,

10, 20, 30, 40, 50 s, 1, 2, 3, 4, 5 and 10 min using the plasma
pencil parameters stated above to determine the dose response
curves. In addition, to determine the long-term effect of LTP,
4

J. Phys. D: Appl. Phys. 45 (2012) 422002

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membrane indicative of dead or dying cells that easily uptake

the dye and appear blue under observation with a microscope,
as evident in figure 3. Prior to assaying the cell viability of
cells at long periods post-treatment, the first sample time was
immediately after plasma treatment of leukemia cells with
LTP. The results at time 0 h post-plasma treatment revealed
that at all doses of plasma the cell counts were equivalent
to the control except for the 10 min treatment time (data not
shown). The 10 min treatment had approximately 95% viable
cells which suggest that if the dose of plasma is high enough,
the effect can be immediate killing of cancer cells. While at
lower doses, there is no immediate cell killing. Surprisingly,
when the cells were sampled at 60 h post-plasma treatment,
statistically significant killing is evident at an exposure of 30 s
(p < 0.05) up to 10 min (p < 0.001). The apparent increase
in cell viability after 36 h with exposures of 1 min or less is
not statistically significant. However, samples analysed at 12,
36 or 60 h indicate that as the dose or exposure of plasma
treatment is increased, the outcome is a highly statistically
significant increase in leukemia cell death after 3 min of LTP
exposure. For example, the results indicate a continued tumour
killing effect beyond the 12 h, with higher doses of plasma,
as evident in the 4 min exposure time which had averaged
percent viability values of 22.3%, 14.2% and 10% at 12 h,
36 h and 60 h post-plasma treatment, respectively, with a highly
statistically significant difference (p < 0.001) when compared
with the control helium-only and non-plasma treated cells. As
expected, the highest cell death was at the highest treatment
time/dose of 10 min of plasma exposure. Even though, lower
doses do not have as great an effect, leukemia cancer cells
continue to die even 60 h post the initial single treatment of
LTP. These data suggest that a single dose of plasma treatment
can have both immediate and long-lasting effects depending
on the dose of the LTP treatment.
The results from this study indicating that there is a dosedependent response in the induction of cell death of cancer
cells correlates with results from other studies which reported
that non-thermal gas plasma has an effect on tumour cell
lines [11, 12]. We hypothesize that the increase in plasma
exposure results in increased concentrations of reactive species
generation. The increased killing effect of higher doses of
LTP are most likely attributed to the higher concentrations
of reactive species that are generated at a given treatment
regime. The single doses of plasma treatment that continue
killing cells at 2.5 days are attributed to long-lasting effects
of some reactive species which trigger internal cell signalling
cascades, while the immediate effect of high doses of LTP
could be attributed to aggressive oxidative action of other
reactive species or possibly some changes to fluid chemistry.
However, further investigations are required to confirm these
assumptions, including the potential effect of the reactive
species on the media. The delayed effect of plasma exposure
on leukemia cells is apparently attributed to the initiation of an
intracellular signalling cascade that progresses to programmed
cell death. This process which takes time is observed at 12 h
post-exposure. Therefore, we conclude that a threshold flux
of reactive species must be generated by the LTP to initiate the
intracellular processes of programmed cell death that result in

statistically significant killing of cancer cells. This process

can be achieved either by increasing the plasma exposure
dose or allowing sufficient time for the reactive species to
initiate an intracellular signalling response in the leukemia
cells. In order to differentiate between the specific mechanisms
of cell death such as apoptosis and necrosis, future experiments
involving DNA fragmentation and flow cytometry will be
pursued.

4. Conclusion
This study revealed that leukemia samples treated with lowtemperature plasma using the plasma pencil have less viable
cells with increasing doses of exposure. These results indicate
that a high dose of LTP has prevented further progression of
CCRF-CEM cell proliferation and is able to induce cell death.
In addition, this study shows that low doses of plasma have a
delayed but statistically significant killing effect on leukemia
cells. Based on these results, low-temperature plasma may be
an innovative alternative therapy to treat cancerous cells. As a
state-of-the-art tool, the plasma pencil can be used to generate
low-temperature plasma for use in inhibiting the progression
of different types of cancers. Future studies will incorporate
testing the efficacy of LTP against other cancerous cell lines
and using electrical engineering and biological techniques
to determine the positive biological effects such as cancer
cell killing with an emphasis on discovering the underlying
mechanisms involved.

Acknowledgments
The authors would like to thank Dr P Hentosh and Dr T Sundin
of the ODU College of Health Sciences for use of their
facilities, cell lines and valuable discussions. We further wish
to thank M A Akman for technical assistance with the plasma
pencil. This work was partly supported by the ODU Office of
Research.

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