Inactivation process of HeLa cell by exposure to
a plasma-treated medium
Takehiko Sato1, Mayo Yokoyama1 and Kohei Johkura2
1
Institute of Fluid Science, Tohoku University, Sendai, Japan
2
Shinshu University School of Medicine, Matsumoto, Japan
Abstract: In this study, we focused on an inactivation process of HeLa cells by
generated chemical species in a culture medium using a plasma flow. The plasma
flow was formed between a platinum needle electrode 0.3 mm in diameter and
the surface of the culture medium with 7.5 kV of positive voltage for 210
seconds. When the cells were incubated continuously in the treated culture
medium, all of them were damaged and underwent necrosis within 13 hours. To
identify the inactivation factor among chemical species, the effect of H2O2 on the
inactivation process of HeLa cells was investigated. The trends of cell survival
rate and morphological change were comparable between them. These results
indicated that H2O2 is one of key factors to inactivate cells.
Keywords: Plasma flow, Inactivation, HeLa cells, Morphological observation,
Survival ratio
1. Introduction
2. Experimental methods
Since a plasma flow has a feature of generating
chemically reactive species, light, heat, electric field,
and shock wave, it has been recently applied to
plasma medicine [1,2] such as a clinical test for
treatment of chronic wounds [3,4], applying to tissue
engineering [5,6] and plasma inactivation for a lowtemperature sterilization [7-13] and preventing
nosocomial infection [14]. As cells/bacteria are
generally covered with water, the plasma-water
system is one of the key phenomena for
understanding the biological response. Various kinds
of chemical species, e.g., NO2, HNO3, O3 and OH,
generated in air dissolve and transport in water
quickly [15], and those chemical stimuli cause
inactivation of bacteria [16]. However, the
mechanism of biological response to plasma flow is
still unclear due to its complexity against many
stimuli generated. In this study, we focused on the
chemical species among the other plasma generated
stimuli to clarify the role of H2O2 for the inactivation
of cell viability by the culture medium exposed to a
plasma flow at atmospheric pressure.
The HeLa cell (Institute of Development, Aging
and Cancer, Tohoku University) was incubated with
Flask for cell culture
Cell
dissociation
Cell
Cell culture
Power
source
Plasma
Culture
medium
Grounded
electrode
Plasma treated culture
medium
Collection of cell
Dissemination of cell
Dissemination into
a 96-well plate
Cell culture in an
incubator for24 hr
Replace with the
plasma treated/
H2O2 added culture
medium
Incubation with
the treated culture
medium
Replace to the
regular culture
medium
H 2O 2
Culture
medium
H2O2 added culture
medium
Incubation
Incubation
Measurement of
absorbance by a
microplate reader
Replace with the
standard culture
with cell count
reagent
Figure 1 Experimental setup and procedure.
Figure 2 Photograph of the culture medium exposing to the
plasma flow.
Figure 4 Spectrum analysis of the plasma flow.
Figure 3 Waveforms of applied voltage and discharge
current.
a regular culture medium which consists of
Minimum 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 in figure 1.
All incubation were performed at 37 ºC with CO2
5% for 24 hr. The time-lapse images of the HeLa
cell were taken by an inverted microscope (Carl
Zeiss, Axio Observer D1).
Plasma flow was generated between a needle
electrode and the surface of culture medium with a
gap of 1.5 mm as shown in figures 1 and 2. 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 ground electrode. The voltage of
+7.5 kV0p with frequency of 5 kHz and duty ratio of
4% was applied to the needle electrode for 210 s.
The waveforms of the applied voltage and the
discharge current were shown in figure 3. A pulse
current was observed corresponding to the plasma
generation. Figure 4 shows the spectrum analysis of
Figure 5 Absorbance intensity of the cell count reagent for
exposed to plasma-treated and H2O2-added culture media.
the plasma flow. The molecular emission lines of
NO and N2 were observed. The H2O2 concentration
was 304 µM in the plasma-treated culture medium
when the exposure time is 210 s.
3. Experimental results
Figure 4 shows the absorbance intensities
obtained with Hela cells 24 hr or 48 hr after 60 minexposure to plasma-treated or H2O2-added culture
media. In the control regular culture, the values at
respective time points were 1.1 and 1.8, showing the
stable increase in cell number. In contrast, they were
remarkably decreased to 0.03 and 0.02 with plasmatreated medium, and to 0.06 and 0.04 with H2O2added medium. Thus, the pattern of decrease in cell
number principally corresponded in both cases.
These findings on living cell number were also
confirmed by morphological observation. Figure 5
shows the time-lapse images for control, plasmatreated and H2O2-added cases from 0 hr to 12 hr.
When the cells were incubated in the regular culture
medium, they divided continuously (arrowheads in
control x20/x63). On the contrary, both cases of
plasma-treated and H2O2-added show a same trend
that the cells started to shrink at around 4hr, formed
a circular bleb at around 6 hr, and died totally by 12
hr (arrows in plasma x20/x63 and H2O2 x20). These
results imply that the H2O2 has an important role in
cellular damage and lethality in the plasma-treated
culture medium.
4. Conclusions
In this study, we have assessed the survival rate
and morphological change of HeLa cells under
incubation with the plasma-treated and H2O2-added
culture media. It was clarified that the inactivation
processes of HeLa cells exposed to the plasmatreated and H2O2-added culture media were
Figure 6 Time-lapse images of control, plasma-treated and H2O2-added cases (x20, differential interference contrast), and of control
and plasma-treated cases (x63, phase difference contrast). The scale bar is 20µm.
comparable.
Acknowledgements
This study was partly supported by the Japan
Society for the Promotion of Science, Grant-in-Aid
for Scientific Research (No. 21246032), and by
Collaborative Research Project of the Institute of
Fluid Science, Tohoku University. We would like to
thank T. Nakajima, Tohoku University for technical
support.
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