# Analysing Mouse Skin Cell Behaviour under a Non-Thermal kHz Plasma Jet
## 4 Discussion  
Experimental results revealed that treating mouse skin cells with an atmospheric pressure kHz argon plasma jet affects cell viability. Our plasma system, used as a direct treatment, is very destructive towards L929 murine fibroblasts, highlighted by the fact that most of the cells are not viable after just 35 s of plasma treatment. To understand what exactly causes this behaviour requires comparison with other studies and plasma systems. However, a comparison of plasma effects on cell viability with other plasma sources is extremely difficult, due to plasma source variances, plasma parameters, types of treated cells, types of plasma treatment and different approaches to the assessment of cell viability. Another problem connected to this research is repeatability, since not all researchers follow the same procedures and protocols. Similarly, researchers may not investigate all aspects needed to explain plasma effects on the treated biological samples. Nevertheless, a comparison of approximately similar plasma systems, presented in Figure 7, highlights our plasma system's toxicity to cells. All selected sources operated in a kHz regime (20 or 40 kHz), except kINPen 09 [16], which operated in the MHz regime and was chosen as the most widely used plasma source for biomedical applications. For ease of reference, different plasma sources were given different symbols in Figure 7. The circle represents kINPen [16] (1.1 MHz, 2-6 kV, Ar--1.9 slm), the triangle represents a jet [7] (40 kHz, 4.9 kV, Ar--4 slm), the square represents a plasma torch [5] (20 kHz, 7 kV, He--5 slm), and the star represents our system. Each cited study focused on slightly different applications, so different types of cell lines were used (mHepR1, A431, HEK293, L929, LNCaP, PC3 and P69). The direct approach is marked as a flat line, and the indirect approach as a dashed line. Results were selected where the distance between plasma and cell medium was 10 mm or less, and viability measured 72 h after treatment. The comparison indicates that our plasma treatment was very aggressive and the most destructive in the shortest treatment time. To explain our jet's destructive nature on the treated cells, we must first look at the gas-phase diagnostics. OES did not show any deviation from expected species present in such plasma. The observed plasma was very stable and produced typical species for atmospheric pressure argon plasma at the beginning and end of the plasma plume (glow region) [17]. The gas temperature (measured as 318 K) is acceptable for in vitro biomedical applications and comparable to other plasma systems (e.g., kINPen 09 operates at 319-323 K [21]). Both time-resolved OES and ICCD images confirmed a regular ionisation process for a kHz-driven plasma source with the formation of pulsed streamers and filaments throughout the waveform [18; 19; 20]. Since the gas-phase plasma parameters seem to be in accordance with other plasma sources used in cell treatment, the chemistry of the plasma-treated cell culture medium was explored. First, we made sure that the cell medium's pH and conductivity did not influence cell viability. Results were stable for all distances and treatment times. Then, we measured concentrations of NO\({}_{2}\)\({}^{-}\) and H\({}_{2}\)O\({}_{2}\) inside the cell medium. Both concentrations were relatively low, and the NO\({}_{2}\)\({}^{-}\) concentration remained almost constant even 72 h after plasma treatment. In contrast, H\({}_{2}\)O\({}_{2}\) concentration gradually decreased and was negligible 72 h after treatment. Comparison of H\({}_{2}\)O\({}_{2}\) production with similar plasma sources is presented in Figure 8. Our results were compared to other sources that operate in the kHz region: 39.5 kHz (marked as a circle [22]), 30 kHz (marked as triangle [9]) and 17 kHz (marked as square [4]). Here, other concentrations are quite high compared to our system.