22G13
Role of hydrogen peroxide for inactivation of cell viability by exposure toplasma-treated culture medium
プラズマ処理培地への暴露による細胞不活性化における過酸化水素の役割
Takehiko Sato1, Mayo Yokoyama1, Kohei Johkura2
佐藤岳彦1，横山茉代1，城倉浩平2
1

Institute of Fluid Science, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan
Department of Histology and Embryology, Shinshu University School of Medicine, 3-1-1 Asahi,
Matsumoto 390-8621, Japan1
東北大学流体科学研究所 〒980-8577 仙台市青葉区片平2-1-1
2
信州大学医学部 〒390-8621 長野県松本市旭3-1-1

2

Recently, medical treatments and sterilization using plasma have come to be known as “plasma medicine” since a
plasma flow is capable of generating various kinds of stimuli such as chemical species, charged particles, heat, light,
shock wave and electric fields. It has been known that a plasma flow has effects on cell viabilities. Here, we focused on
an inactivation effect of chemical species generated by a plasma flow and aimed at identifying a key factor ofinactivation of cell viability among stable chemical species. To identify the chemical species of the inactivation factor,
we focused on the effect of H2O2 in plasma-treated culture medium because it is generated in the culture medium and it
is also chemically stable compared with free radicals generated by the plasma flow. To elucidate the significance ofH2O2, we assessed the differences in the effects of plasma-treated medium and H2O2-added medium against inactivationof HeLa cell viability. These two media showed comparable effects on HeLa cells and the results supported that amongchemical species generated in a plasma-treated culture medium, H2O2 is one of main factors responsible for inactivationof HeLa cell viability.

1. Introduction
A plasma flow has been recently applied toplasma medicine since it is easily capable ofgenerating chemically reactive species, light, heat,electric field, and shock wave [1,2]. Plasma
inactivation for a low-temperature sterilization [3]have been reported as the outcome of researches in
the plasma medicine field.
Since cells/bacteria are generally covered withwater, the plasma-water system is one of the key
phenomena for understanding the biologicalresponse. Various kinds of chemical species, e.g.,
NO2, HNO3, O3 and OH, generated in air dissolve
and transport in water quickly and decreased pH
value [4, 5]. However, the mechanism of biologicalresponse to plasma flow is still unclear due to its
complexity against many stimuli generated.
To identify the specific chemical species which
has an inactivation effect on cell viability, we
focused on the H2O2 among the other species
generated by plasma.
2. Experimental methods
The HeLa cell (Institute of Development, Agingand Cancer, Tohoku University) was incubated witha regular culture medium which consists ofMinimum Essential Medium with addition of 10%
Fetal Bovine Serum and Penicillin-Streptomycin(Penicillin 100u/ml; streptomycin 100μg/ml). The
procedure to measure the absorbance intensity

which indicates the number of cells was shown inFig. 1. All incubation were performed at 37 ºC with
CO2 5% for 24 hr. The time-lapse images of theHeLa cell were taken by an inverted microscope
Cell Flask for cell culture Cell culture in
an incubator

Cell abrasion
Collection of cellPowersourcePlasmaCultureGroundedelectrodePlasma treated culture
H2O2Culture
H2O2 added culture

Dessemination of cell
Dissemination intoa 96‐well plate
Cell culture in an
incubator for24 hr
Replace to theplasma treated/
H2O2 added culture
Incubation withthe treated culture
Replace to thestandard culture
Addition of cellcounting reagent
Incubation
Measurement ofabsorbance by
micro plate reader

Fig. 1 Experimental setup and procedure.

3. Experimental results and discussion
Figure 2 shows the cell survival rates shown by
the absorption intensities in the cases of regular(control), plasma-treated and H2O2-added culture
media.
Theabsorption
intensities
withplasma-treated and H2O2-added media decreased
with increase of the exposure time and the cells
were not alive in the case of 120 min though thecontrol did not decrease. The trend of survival rate
against the exposure time for the plasma-treated andH2O2-added cases was similar [6].
In order to assess the biological reactions of cells
to plasma-treated culture medium, we carried out
comprehensive gene expression analysis (Whole
Human Genome Microarray Kit, Agilent
Technologies) and obtained the comprehensive
gene expression profiles. Figure 3 shows the scatterplot of the gene expression intensities made by aGeneSpring software (Agilent Technologies). If theexpression intensities of a given gene are the samevalues between plasma and H2O2 cases, the plot islocated on the central diagonal line, and if not, it
deviates from the line. When gene expression levels
in plasma-treated medium were compared withthose in H2O2-added medium, most of plots for highexpression genes were located on the diagonal line,
suggesting a comparable cellular response [6].
4. Conclusions
In this study, we have assessed the survival rate
and the comprehensive gene expression intensities.
It was clarified that H2O2 is the inactivation factor
of HeLa cell viability among chemical species
generated by exposure to the plasma flow.
This study was partly supported by the Japan
Society for the Promotion of Science, Grant-in-Aidfor Scientific Research (No. 21246032), and by
Collaborative Research Project of the Institute ofFluid Science, Tohoku University. We would like tothank T. Nakajima, Tohoku University for technicalsupport.

1.4
Absorption intensity (a.u.)(Carl Zeiss, Axio Observer D1).
Plasma flow was generated between a needle
electrode and the surface of culture medium with agap of 1.5 mm. The electrode is made of platinum,0.3 mm in diameter. The culture medium of 1.0 ml
in a microtube of 1.5 ml was set in a groundelectrode. The voltage of +7.5 kV0p with frequencyof 5 kHz and duty ratio of 4% was applied to theneedle electrode for 210 s. The power consumption
was 7.1 W. The H2O2 concentration was 304 µM in
the plasma-treated culture medium when theexposure time is 210 s.

1.21Control
Plasma-treated
H2O2-added

0.8
0.6
0.4
0.2010 min

30 min60 min
120 min
Exposure time to media

Fig. 2 Cell survival rate shown by the absorption
intensity versus exposure time to media of the regular(control), plasma-treated and H2O2-added.

Fig. 3 Scatter plot of the intensities corresponding
to each gene under plasma-treated case versus
H2O2-added case. The values in both vertical andhorizontal axes are a common logarithm (Log10).
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