## 4 Discussion
Previous studies have demonstrated that the exposure of different culture media to CAP can induce modifications of their components [72; 73; 74]. Moreover, while [PERSON] et al. showed that the presence of pyruvate in PAM inhibits its cytotoxic activity, [PERSON] et al. demonstrated that the only constituent element in [PERSON]'s lactate solution whose exposure to plasma gives the entire solution anti-tumor characteristics is L-sodium lactate [75; 76]. The possible reactivity between CAP and the multitude of constituent elements of the RPMI medium, in contrast to the simple PBS solution, may be the cause of the higher cytotoxicity observed in this study when using PAM-RPMI. This assumption should be verified in the future by analyzing which element or combination of elements following CAP exposure, in the same concentration or concentrations as in the evaluated RPMI 1640, gain cytotoxic properties similar to PAM-RPMI. Therefore, according to our results, RPMI 1640 was selected as the solution of choice for producing PAM in the subsequent stages of this study. An important parameter in indirect CAP treatment is the interaction between the PAM and the target material (i.e., HOS cells), also called treatment time. According to the specialized literature, in previous studies different treatment times were used, starting from 10 min, up to 72 h [3; 9; 60; 75; 77; 78; 79; 80; 81; 82; 83; 84]. According to [PERSON] et al., the indirect method typically requires significantly longer treatment time than direct treatments with CAP in order to achieve the same level of efficiency [85]. As a result, most studies using the indirect method for OS employ treatment durations of 2 h or more, resulting in bone tumor cell viability decreasing between 80% and 0% at 24, 48, or 72 h post-treatment [9; 86; 87; 88]. In these cases, the effectiveness of the treatments depended on various factors, including exposure and treatment times, PAM solution type, cell type, and also the time between treatment and evaluation. Our study found that even with a short 30-min PAM treatment, we were able to achieve a viability decrease in HOS cells down to 28% in just 2 h after the treatment. These results are comparable to the results of other studies that used the direct method [12; 89; 90; 91; 92], which is generally accepted as the most effective method for reducing cell viability with CAP treatment. However, our study achieved these results with a shorter exposure and treatment times than those typically used in the indirect method. Observing that the color of the RPMI 1640 medium (containing phenol red) changed gradually during the exposure to CAP discharges, depending on the T\({}_{\rm exp}\) used, the subsequent stage of our investigation was to assess the pH of PAM-RPMI. The results show that the exposure of the RPMI 1640 to CAP determines its acidification, in a dose-dependent manner. Previous studies have observed similar [78; 93] or even stronger acidification [3; 94] of solutions exposed to CAP. However, [PERSON] et al. and [PERSON] et al. were able to produce PAM without any alterations to the pH levels. The main difference in their approach was the addition of FBS to the solution that was exposed to CAP, either before or after the exposure [9; 81]. According to [PERSON] et al., the pH reduction observed following CAP exposure is a result of increased hydrogen ion concentration [3]. After observing the acidification of PAM-RPMI, our objective was to determine the impact of pH variations caused by treatment on the viability of HOS cells indirectly exposed to CAP. Specifically, we aimed to differentiate whether the decrease in cell viability was due to the effects of CAP treatment or to the acidification of the treatment solution. Thus, we concluded that only the low pH RPMI 1640 does not influence the viability of HOS cells, the cytotoxic effects being, therefore, determined by the exposure of RPMI 1640 to CAP. The results support [PERSON] et al.