Plasma controlled cell migration.
Olga Volotskova, Mary Ann Stepp, Michael Keidar
The George Washington University
Abstract: Recent breakthrough research in the area of cold atmospheric plasmas demonstrated great potential in
various areas including medicine and biology, providing tool for living tissue treatment. Our recent work was
directed at understanding the mechanism by which the plasma jet alters the cell migration and influences its
adhesion. This report is focused on the studying of cold atmospheric plasma jet interaction with two types of cells
fibroblast (dermal) and corneal (epithelial) cells. The ability of plasma to reduce the cell migration rates is
demonstrated. It is shown that migration rates of the treated cells change locally (localization effect of the plasma
treatment). The cells response to the duration of plasma treatment was studied and migration rate sensitivity on
time together with the threshold of the migration rate change was determined. Data showed that the ability of
plasma to reduce cell migration rates increases as a function of treatment time with a maximum of ~30-40% and
that this affect persists for at least 33 hours after plasma treatment. Also two extreme states of the cells: integrin
activated by MnCl2 and serum starved cells were studied and compared with plasma treatment.
Keywords: Cold atmospheric plasma jet, Cell migration, Cell Adhesions, Integrins, Wound
healing
1. Introduction
Migration of the tissue cells plays an important role
in many physiological and pathological processes,
including embryonic development, wound repair,
angiogenesis, and the metastasis of tumor cells.
Various signaling pathways control these processes.
Integrins are a major family of metazoan cellsurface-adhesion receptors playing a key role in the
signaling and mechanotransduction mechanisms:
their function is maintaining cell adhesion, tissue
integrity, cell migration, and differentiation.
Integrins consists of two non-covalently associated
subunits α and β subunits, each of which is a singlepass type I transmembrane protein.1 Recently it was
found that cold atmospheric plasma jets can slow
down the cell migration2 and change integrin
expression on the cell surface, i.e. treatment of cells
with plasma jet resulted in a decrease of β1 and αvintegrins.3 In addition, the localization effect of cold
plasma treatment on the cell migration was shown
(see Figure 1). It appears that the plasma effect is
determined by the size of plasma jet and does not
alter cell migration outside the treated area. And that
it has not only spatial but temporal dependence as
well since this effect persists at least for 33 hours.4
Despite the progress made in studying of cold
plasma interaction with the living tissue, question
remain: what are causes the motility and cell
adhesion of the cells change?
Figure 1. A. The experimental set up for spatial distribution of
the cell migration rates for single well is shown. B. The
dependence of cell migration rates vs. distance from the center
of treated zone: for WTDF 3.
Moreover, this question is directly related to the
problem of the wound healing. And our recent work
was directed at understanding the mechanism by
which the plasma jet alters the cell migration and
influences its adhesion. This report is focused on the
studying of cold atmospheric plasma jet interaction
with two types of cells fibroblast (dermal) and
corneal (epithelial) cells.
2. Methods and Materials
Cell culture. Wild type tertiary mouse fibroblast
cells (WTDF 3) were cultured in the Dulbecco’s
Modified Eagle Medium (Invitrogen Corp.) enriched
with 5% serum, 1% NEAA, 1% L-glutanine, 1%
Pen-Strep. Diluted cells (30% confluence) were
plated in multi-well plates or double-well glass
slides and treated with plasma on the 3rd day in
culture. Human corneal limbal epithelial cell line
(HCLE, Dr. Ilene Gipson, Harvard Medical School)
was grown in HCLE medium (500ml Keratinocyte
Serum Free Medium with added 1.2ml Bovine
Pituitary Supplement, 4ul Epidermal Growth Factor,
0.3mM CaCl2 and 5ml Pen-Strep, Invitrogen Corp.).
HCLE cells were used on the 2nd day of culture with
confluence of around 30%. During the experiments
plates with cells were kept on the heating plate
(Boekel scientific, model 240000) to maintain the
media temperature at 37°C. The serum starvation
was performed on WTDF 3 cells: DMEM with 1%
serum was added in 24 hours before the experiment.
MnCl2 treatment of WTDF 3 was performed
immediately after the experiment (dilution 1:2000,
DMEM 5%).
Plasma jet. The output voltage is around 4.5-5kV,
the frequency is ~13kHz, the helium flow rate ~1112l/min. The distance between the jet outlet and
culture plate/slide was always kept ~ 20mm. The
depth of the media was maintained ~ 2mm. Dermal
fibroblasts and epithelial cells were prepared and
equivalent numbers plated onto culture plastic (for
time-lapse
studies)
or
glass
slide
(for
immunostaining) and maintained at in DMEM
medium supplemented with 5% serum and HighCalcium HCLE medium. The fresh media was added
to all the cells right after the experiment.
Time-lapse studies. Further time-lapse studies were
performed on an Olympus IX81 research microscope
(Olympus America, Center Valley, PA) equipped
with a Proscan motorized stage (Prior Scientific
Instruments Ltd., Rockland, MA) and placed in a
temperature and CO2 controlled chamber (LiveCell
Incubation System, Neue Biosciences, Camp Hill,
PA). Using relief-contrast optics, images were taken
every 10 minutes for 16 hours 40 minutes (100
images). Images were transferred to a workstation
equipped with Metamorph image analysis software
(Molecular Devices Corporations, Chicago, IL)
where velocities of 10 cells were calculated using
the track cell module in each tracked location. More
detailed description can be found elsewhere5.
Fluorescent studies. Cells were fixed in 4%
formaldehyde. The antibodies listed below for
biochemical analyses and microscopic localization
of proteins were used. Total b1 integrin was shown
by means of monoclonal biotin-conjugated hamster
CD 29 (integrin b1 chain), dilution 1:200 (BD
Bioscience, cat#55504). For activated b1 integrin
purified rat CD 29, clone 9EG7 was used, dilution
1:200 (BD Bioscience, cat#553715). Nuclei were
visualized using DAPI, dilution 1:2000 (D21490;
Invitrogen Corp.). Images were acquired at !40
magnification using a Nikon Fluorescent microscope
equipped with a RT-Slider SPOT Camera (Melville,
NY). Adobe Photoshop 7.0 was used to manage
images. For quantification analysis Image Pro Plus,
version 6.2 software (Media Cybernetics Inc.) was
used.
3. Results.
Presented in Figure 2A are relief contrast images of
the tracks of the HCLE cells with and without
plasma treatment. In the Figure 2B the cell migration
is assessed as a function of the duration of plasma
treatment: WTDF 3 (blue) and HCLE (red). In both
cases cells show decrease in the migration rates as
the duration of the plasma treatment increases,
however there’s a significant difference in the
threshold of the change of the migration rates.
plasma treated (100 seconds) with MnCl2 treatment
(integrin activation condition1) - red, serum
starvation (quiescent condition) - green and without
additional treatment applied - blue. In the case with
no additional treatment applied there’s a ~30-40 %
drop in the migration rates of the cells between
controls (no treatment and only helium treated) and
plasma treated cells. However, if the additional
conditions were applied no any changes in the
migration rates were observed. Another interesting
fact is that the decrease of the cells migration treated
with MnCl2 and serum starvations in the case of
controls show a similar 30-40% decrease, and
there’s no difference in the migration rates of the
cells treated with plasma.
Figure 2. A. Relief contrast images of the HCLE cells. B.
Migration rate as a function of the length of the plasma
treatment shown (WTDF 3 – blue and HCLE – red). C.
Persistence as a function of the length of the plasma treatment
(WTDF 3 – blue and HCLE – red).
Dermal fibroblast cells show a maximum drop in the
migration rate change at around ~ 40 seconds, but
the impact of plasma on the migration of epithelial
cells is more graded, having its threshold of change
around ~ 100 seconds. Interestingly that the
maximum change of the migration rates in both
cases is around ~ 30-40%. Further, on the threshold
was achieved there are no significant variations in
the cell migration rate. Figure 2C shows the
persistence of the cell motion as a function of the
duration of the plasma treatment. The persistence
was measured as a ratio between net and total
displacements and, thus, represents the directionality
of the cells motion. Neither
dermal fibroblasts,
neither corneal epithelial cells show significant
changes in the cell migration as the length of the
plasma treatment time increases.
Figure 3 shows the migration rates of the WTDF 3
cells: controls (not treated, only helium treated) and
F
igure 3. The migration rates of the WTDF 3 cells: control (no
plasma, no helium treated), only helium treated and plasma (100
seconds) treated are shown. No additional treatments applied –
blue; treated with MnCl2 – red; and serum starved – green.
Figure 4 shows the immunofluorescence studies of
the activation state of the b1 integrin, i.e. its epitope
recognized by 9EG7 antibody will be expressed only
when the β1 is activated. Total β1 is shown in green
and 9EG7 – in red. The untreated cells (control) and
plasma (plasma 100sec) treated cells are shown in
the Figures 4A and 4B with and without additional
MnCl2 treatment. The activated β1 integrin is
present more in the cells treated with plasma rather
than in the untreated cells, but there are no
significant changes in the 9EG7 expression in the
case of MnCl2 treatment. Also the expression of
9EG7 in plasma treated, MnCl2 treated and plasma
and MnCl2 treated cells has a trend to appear more
at the periphery (shown with arrows).
atmospheric plasma jet is seen for both: WTDF 3
and HCLE cells, and the maximal reduction of the
migration rate is around ~30-40%. However, it takes
longer plasma treatment times for HCLE cells to
achieve the maximum reduction of migration rate.
No significant changes in the persistence of the cells
motion (WTDF 3 and HCLE) were found. No
changes in the migration rates of MnCl2 or serum
starved fibroblast cells, i.e. cells with activated
integrins (MnCl2 and serum starvation induce
integrin activation)1,6, treated with plasma were
found. Also the maximum drop in the migration rate
is the same (30-40%) in the case of the plasma
treatment and integrin activation of the fibroblast.
Interestingly, that this (30-40%) difference is a kind
of the threshold of the cells velocity change. The
fluorescence analysis showed the increase in the
expression of the activated β1 integrin after the
plasma treatment of WTDF 3 cells. From this data, it
can be concluded that the integrin activation is one
of the mechanisms by which cold plasma jet
interacts with the cells.
References
[1] R.O. Hynes, Cell, 110, 673–687 (2002)
[2] A. Shashurin et al., Appl. Phys. Let. 93, 1815013 (2008)
[3] A. Shashurin et al., Plasma Process. Polym., 7,
294–300 (2010)
[4] O. Volotskova et al., Plasma Medicine, 1,1, 8592 (2011)
[5] R.A. Jurjus et al., Wound Rep. Reg. 16, 649–660
(2008)
[6] M. A. Stepp et al., in progress.
Figure 4. The WTDF 3 cells stained against total β1 – green,
9EG7 – red and DAPI – blue. A. Control and plasma treated
(100 seconds) cells with no additional treatment. B. Control and
plasma treated cells MnCl2 treated.
4. Conclusions.
Here it has been shown that the decrease in cell
migration rates can be induced by the cold