## 4 Discussion  
### Fish Cells Respond Differently to Plasma Treatment than Mammalian Cells  
As a first step on the way to a CAP-mediated therapy of farm fish, we wanted to clarify how piscine cells react to CAP treatment, as this was previously unknown. For this reason, we performed cell viability assays to evaluate the individual responsiveness of gill epithelial cells and fibroblast-like cells from rainbow trout and liver epithelial cells of the three species rainbow trout, mouse, and human. The respective cells were chosen because the use of primary non-cancerous human cells in cell culture often requires highly supplemented media adapted to the specific needs of that particular cell line. Considering that our main experimental principle was to use an identical medium to deliver an identical cocktail of plasma-generated reactive species to the different cell lines, the use of cells requiring a specific medium had to be avoided. Therefore, only cells that could be cultured in the same medium were selected. This allowed us to draw conclusions for the newly used fish cells compared to the already published mammalian cells. Our aim was to provide an objective comparison to place the fish cells in this known context. The results of an experiment depend, among other variables, on the careful choice of the cell number. To allow for comparisons across different cell lines, the design of an experiment should involve tuned cell numbers. In addition to the adjustment of the cell number, proliferation rates, metabolic status and cell migration effects should be taken into account [32; 43]. In line with this, we cultured the piscine cells and the mammalian cells at equal cell numbers in identical medium and found that the fish cells reacted much more sensitively to CAP treatment than the mammalian cells. Subsequently, we changed the experimental conditions with regard to cell density to ensure a similar degree of confluency prior to the plasma treatment. This took into account the size of the individual cells as well as their proliferation rate. The results did not provide clear indications for the interdependence of confluency and cellular sensitivity to CAP. While one piscine cell line (RTgill-W1) showed a higher robustness to PAM after the careful adjustment of the optimal confluency, the sensitivity of another confluent cell line from fish (OMYsd) remained almost unchanged. These differences could be due to specific structural and functional adaptations, as the cell lines were derived from different tissues of rainbow trout (liver, skin and gill) and display different morphological characteristics (see Figure 3). The serum used to supplement the cell culture media may also have influenced the sensitivity of the individual cell lines towards PAM. We used serum from fetal calves  
Figure 7: Cell viability of plasma-treated cells cultured in DMEM without pyruvate compared with DMEM with pyruvate (1 mM). Fish cells OMYleb and OMYsd react much more sensitively to PAM without pyruvate, while the other cell lines are less affected (\(n=3\)). (FCS), which has been used as a universal supplement for more than six decades [44] to culture approximately 95% of all cell lines including insect cells [45]. Essentially, the supplementation of cell media with FCS ensures the supply with growth factors, which are evolutionarily well conserved [46]. However, the exact composition of FCS has not yet been clarified [47], which is also due to the different origins of the cattle stocks. Therefore, we used a serum for our experiments that is composed in a lot-independent manner (FCS Superior, Biochrom GmbH). Since serum contains thousands of different metabolites [48], it is not yet possible to assess whether and how specific serum factors differentially affect cellular signaling cascades and survival pathways in fish or mammals.