22nd International Symposium on Plasma Chemistry
July 5-10, 2015; Antwerp, Belgium
Dynamic behaviour of glioblastoma cells in plasma-activated medium
H. Tanaka1, M. Mizuno1, K. Ishikawa1, K. Takeda1, H. Hashizume1, K. Nakamura1, F. Utsumi1, H. Kajiyama1,
H. Kano2, Y. Okazaki1, S. Toyokuni1, S. Maruyama1, T. Adachi3, H. Kaneko1, H. Terasaki1, Y. Kodera1, F. Kikkawa1
and M. Hori1
1
Nagoya University, JP- 464-8603 Nagoya, Japan
2
NU-Eco Engineering
3
Gifu Pharmaceutical University
Abstract: Recently, we have reported that Plasma-activated medium (PAM) has antitumor effects on several cancers, and an anti-angiogenic effect on choroidal
neovascularization. To gain insights into interactions between PAM and cells, we
conducted time-lapse microscopy of glioblastoma cells. Time-lapse images revealed the
dynamic behaviour of PAM-treated glioblastoma cells.
Keywords: plasma-activated medium, cancer, time-lapse imaging
1. Introduction
Plasma medicine is an emerging interdisciplinary field
that involves not only plasma physicists and medical
doctors but also biologists, chemists, and other experts
who would like to understand the phenomena mediated by
non-thermal plasma and apply for medicine. In these
days, non-thermal plasma has been applied for wound
healing, hemostasis, sterilization of medical instruments,
and so on [1-3].
In the field of plasma medicine, plasma irradiated
medium, what we call plasma-activated medium (PAM),
is attracting interests because it extends application over a
wide range. We have shown potential antitumor effects to
glioblastoma, ovarian cancers, and gastric cancers by
PAM [4-7]. We also have recently reported that PAM
might be a new therapeutic method for age-related
macular degeneration [8].
Plasma interacts with nitrogen, oxygen, components in
solution, and water in gas phase and liquid phase to
produce reactive nitrogen and oxygen species in liquid.
Although plasma chemistry in these processes remains to
be elucidated, the physiological outputs such as PAMmediated apoptosis and different sensitivities to PAM (ex.
glioblastoma vs astrocyte) suggest that it is important to
understand interactions between PAM and cells. Based
on the understanding of intracellular molecular
mechanisms of PAM-mediated apoptosis [5, 9], we might
be able to propose how to produce effective and safe
PAM for future clinical applications.
2. Materials and Methods
U251SP cells (human glioblastoma brain tumor cell
line) were grown in Dulbecco’s modified Eagle’s medium
(DMEM; Sigma-Aldrich, MO, USA) supplemented with
10% fetal bovine serum (FBS),100 units/ml penicillin,
and 100 µg/ml streptomycin (P/S) under an atmosphere of
5% CO 2 / 95% air at 37 °C.
Plasma-activated medium was prepared as shown in
Fig. 1. The flow rate of argon gas was set at 2.0 standard
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litter/minute (slm). Three mL of DMEM medium without
FBS and P/S in a 6-well plate was treated with plasma
(distance between the plasma head and the medium
surface was 13 mm) for 5 minutes. Then, the plasma
irradiated medium (PAM) was replaced with the medium
on cultured cells.
Fig. 1. Three mL of medium in a 6-well plate was treated
with plasma (the distance between plasma source head
and the medium surface was 13 mm).
For time-lapse imaging, cells were plated on a glass
base dish. On the following day, images were collected at
5 min intervals using a microscope with an incubator
(OLYMPUS LCV110) immediately after PAM was
replaced on the glass dish.
3. Results and Discussion
We investigated the morphological effects of PAM
against glioblastoma brain tumor cells. PAM treated cells
were observed by microscope 4 hours after cells were
treated with PAM (Fig. 2).
1
Glioblastoma cells have flat shape on the glass base
dish in untreated medium, form lamellipodia, and move
using cytoskeletal structures on the dish. Those cells
formed protrusions within 60 minutes after the cells were
treated with PAM. The cells formed some small balloons
210 minutes after PAM treatment, and those balloons
grew and fused with other balloons. The cells finally
(5~6 hours after PAM treatment) formed a big balloon.
The formation of balloon of each cell was observed at the
same time (3~5 hours after PAM treatment) (Fig. 3).
Fig. 2. Glioblastoma cells were treated with PAM, and
the morphology was observed by a microscope. PAM
induced balloon-like morphology (black arrows) on
glioblastoma cells. Cleaved Caspase3/7 (white arrows)
was detected in these cells. Scale bar represents 50 µm.
Most of PAM-treated cells have shown balloon-like
morphology which is characteristic to apoptosis. To
investigate whether cells underwent apoptosis, we
labelled cells with an apoptotic marker, cleaved
Caspase3/7. The apoptotic signals were detected in cells
that bear balloon-like morphology.
Morphological information gives us valuable insights
into cellular interactions of surrounding environments.
Cell surface is called plasma membrane which consists of
lipid bilayer and several transmembrane proteins such as
receptors that transduce intracellular signalling, ion
channel molecules, and proteins that interact with
extracellular matrix and neighbour cells. Cells respond to
environmental stimuli and show physiological outputs
such as apoptosis and morphological changes.
Cytoskeleton is located under the plasma membrane, and
cellular morphology is regulated by cytoskeleton
regulatory proteins.
Many researchers in plasma medicine have reported
that non-thermal plasma induced cell death including
apoptosis and necrosis. However, physiological outputs
induced by plasma are complicated, and should be
characterized based on cellular biological analyses.
To understand interactions between PAM and cells in
terms of cellular morphology, we conducted time-lapse
microscopy.
One hundred thousands of glioblastoma cells were
plated on glass base dish. On the following day, the
plasma-activated medium was prepared and replaced as
described in Materials and Methods. Images were taken
at 5 minutes intervals.
2
Fig. 3. Glioblastoma cells were treated with PAM, and
the time-lapse images of glioblastoma cells were taken at
5 minutes intervals. Black arrows indicate balloon-like
morphology. Scale bar represents 50 µm.
4. Conclusion
It has been demonstrated that PAM might be powerful
therapeutic methods for cancer therapy and age-related
macular degeneration. In this study, we have shown that
time-lapse imaging of PAM-treated cells might provide
insights into interactions between cells and PAM.
Characterization of PAM based on time-lapse imaging
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would help us to elucidate mechanisms of physiological
outputs induced by PAM.
5. Acknowledgements
This work was partly supported by a Grant-in-Aid for
Scientific Research on Innovative Areas “Plasma Medical
Innovation” Grant No. 24108002 and 24108008, and a
Grant-in-Aid for Young Scientists (B) “Analyses of
intracellular molecular mechanisms of cell death of
cancer cells by plasma” Grant No. 25870307 from the
Ministry of Education, Culture, Sports, Science and
Technology of Japan.
[9]
T. Adachi, H. Tanaka, S. Nonomura, H. Hara,
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induces A549 cell injury via a spiral apoptotic
cascade involving the mitochondrial-nuclear
network". Free Radic. Bio. Med., 79C, 28-44
(2014)
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