Title: Indirect plasma treatment of SAOS-2 and NHDF cells by surface dielectric
barrier discharge
Type: Poster, 7) Plasma Medicine
Authors: Satomi Tajima1, Roberto Gristina2, Paolo Ambrico2, Daniela Pignatelli3, Kai
Masur4, Klaus-Dieter Weltmann4, Thomas von Woedtke4, Hirotaka Toyoda1, Masaru
Hori1, Pietro Favia3
1. Plasma Nanotechnology Research Center, Graduate School of Engineering, Nagoya
University, Furo-cho, Chikusa-ku, Nagoya-city, Aichi, Japan, 464-8603
2. IMIP, CNR c/o Università di Bari, Via Orabona 4, 70126, Bari, Italy.
3. Dipartimento di Chimica, Università degli Studi di Bari, Via Orabona 4, 70126, Bari,
Italy.
4. Leibniz Institute for Plasma Science and Technology (INP), Greifswald, Germany.
Research aims/questions:
Cold plasma discharges are promising therapeutic tools for would healing,
blood coagulation, and treatment of cancer cells [1]. . In order to understand the
mechanisms underlying the different interaction between plasma and eukaryotic cells, in
vitro experiments with cell lines represent a very powerful tool. From our previous
study, we found that the significant difference in cell liability and proliferation behavior
of Sarcoma Osteoblasts (SAOS-2) and Normal Human Dermal Fibroblasts (NHDF)
were observed [2]. In this study, we investigated the change in cell liability and
morphology of those cells which were plasma treated indirectly where complete
medium was treated with a surface dielectric barrier discharge (surface-DBD).
Methodology:
50,000 SAOS-2 and NHDF cells were seeded on 60 mm polystyrene Petri dish
and incubated for at least 20 hours.
A surface-DBD apparatus was used to modify 3 mL of DMEM medium
supplemented with 10% FBS (Sigma Aldrich) to be added to cells. The detail
description of this apparatus is described elsewhere [2]. The rectangular waveform with
the on time of 970 ms and the off time of 30 ms were initiated by the pulse generator
(Nuova Elettronica) and introduced into the power supply (PVM/DDR plasma resonant
driver, Information Unlimited) which can generate sinusoidal waves with the frequency
of 20.7 kHz and the peak-to-peak voltage of 12 kV. The distance between the patterned
electrode and the bottom of the 60 mm polystyrene Petri dish was adjusted to 5 mm.
Plasma treatment time was set at 15s, 60s and 120s for Saos2 cells and 15s, 30s
and 60s for NHDF cells since previous experiments have shown that the latter were
more sensitive to the plasma treatment. Plasma treated medium was added to cells for 1
h and replaced to the complete medium and incubated for 23 h and 71 h. Total
incubation time of the cell after the medium plasma treatment was 24 h and 72 h. The
cell liability and proliferation was analyzed using MTT assay by calculating the
difference in absorbance at 570 and 690 nm. Cell morphology is analyzed by means of
Coomassie Blue, Actin filament observation, and DAPI staining.
Results
Figure 1 shows the absorbance values calculated from MTT assay which was
prepared 24 h and 72 h after the cell was incubated in the plasma-treated medium
followed by the complete medium. Both cells show lower cell liability as the plasma
treatment time of the medium increased. When medium was treated for 15 s and 30s,
the plasma modification of cell medium do not inhibit cell proliferation for both cells.
When medium was treated for 60 s, NHDF lost cell liability whereas Saos-2 cell
survived after the 72 h incubation. Liability Saos-2 cell was lost significantly after the
medium was plasma-treated for 120 s and incubated for 72 h.
Figure 1: Analysis of growth of Saos2 and NHDF cells by means of MTT essay.
Significant reduction in cell spreading area, actin filament, and the size of the nuclei
were observed after the medium was plasma-treated for 60 s for Saos-2 cells. The
reactive species in the medium that plays a key role in modifying above cell behavior is
under investigation.
Conclusions
We evaluated the change in cell liability and cell morphology after Saos-2 and NHDF
cells were incubated in plasma-treated medium for 1 h followed by incubating in the
complete medium for 23 and 72 h. The increase of the plasma-process time of the
medium would affect the cell liability, proliferation, and morphology for both cells.
[1] Plasma Processes & Polymers 5(7), 2008; Special Issue on Plasma Medicine, organized by
A. Fridman and M. Laroussi.
[2] P. Favia, D. Pignatelli, G. Dilecce, B. R. Pistillo, M. Nardulli and R. Gristina.. MRS
Proceedings, 2012 1469.