## 4 Discussion
### Fish Cells Respond Differently to Plasma Treatment than Mammalian Cells
Although we tried to keep the cultivation conditions as similar as possible for the five cell lines investigated, the cultivation temperature was a key parameter that had to be kept within the tolerance range of the donor species rainbow trout, mouse, and human. Rainbow trout are explicitly cold-water fish and grow at temperatures between 8 \({}^{\circ}\)C and 22 \({}^{\circ}\)C [49]. Consequently, we cultured the fish cells at 20 \({}^{\circ}\)C and not at 37 \({}^{\circ}\)C like their mammalian counterparts. Previous studies revealed that culturing fish cells at 37 \({}^{\circ}\)C produces a pronounced heat-stress response [50]. Nonetheless, it is well established that temperature determines the activity of metabolic processes. The fact that fish cells were cultivated in a 17 \({}^{\circ}\)C colder environment is expected to have influenced the cellular metabolism. The van-'t-Hoff rule states that temperature reduction significantly slows down chemical processes [51; 52]. In addition, temperature defines the fluidity of lipid bilayers and therefore the membrane competency including ion transport, enzyme function and the formation of signalling complexes [53]. In the present study, we observed that fish cells reacted more sensitively to oxygen radicals compared with mammalian cells. We can only speculate that distinct temperature-dependent kinetics of the involved anti-oxidative enzymes and the relevant signalling cascades may have influenced the fate of the mammalian and fish cells. In summary, in all experimental setups, the piscine cell lines (OMYleb, OMYsd and RTgill-W1) were the most sensitive to plasma-treated liquids. Therefore, we can conclude that the treatment of fish cells requires carefully adjusted plasma treatment times. As a first step, we performed some experiments on the wound healing potential under our experimental conditions (Figure S1). However, since we found that the sensitivity of the fish cells was very high in our experiments, we were unable to measure any positive effect on wound healing, even with the shortest treatment times. The establishment of suitable plasma treatment regimes in terms of reactive species composition and application protocols will be a task for future research. The Choice of Culture Conditions Is Highly Relevant as Changes in the Antioxidant Capacity Make a Difference  
The effects of CAP are mediated via the cell culture medium. Therefore, we wanted to address the question to what extent a change in culture conditions modulates the cellular response to CAP treatment. The optimal composition of the cell culture medium has been tailored for the broad range of secondary cell lines and primary culture. Consequently, many different media are commercially available with DMEM (Dulbecco's modified Eagle medium)/EMEMEM (Eagle's minimum essential medium) and RPMI (Roswell Park Memorial Institute) as the most frequently used growth media [54]. Furthermore, various additives such as serum, amino acids, D-glucose or phenol red are added in different concentrations. All of these components may interfere with the eventual effects of physical plasma. We studied the influence of two different media on the PAM effects measured on the five cell lines. Since L-15 medium is the common medium for the culture of salmonid cells [41; 42], we used it for our comparative experiments. The direct comparison to the cultivation and treatment in PAM generated from DMEM revealed that the fish cells were more sensitive to PAM from L-15 medium treated with CAP for short periods and more tolerant to PAM from L-15 treated with CAP for a longer period. These diverging effects are likely to result from the different composition of the media. The concentration of potentially anti-oxidative compounds might be of crucial importance. We stored the PAM for 24 h before adding it to the cells because this allowed us to exclude the possibility that the observed effects were due to high hydrogen peroxide concentrations or oxygen depletion during plasma treatment.