Cold atmospheric pressure plasma (CAP) has emerged as a promising therapeutic modality in wound healing, with multiple devices now certified for clinical use. However, the constructive and functional diversity of CAP technologies poses significant challenges for cross-device comparison in preclinical in vitro studies. In this study, we evaluated how device-specific parameters and experimental conditions influence cytotoxic outcomes across different CAP technologies. Using L929, GM00637, and HaCaT cells, we compared direct treatment with a plasma jet (kINPen® MED) and a surface micro-discharge device (plasma care®), revealing significant differences in the reduction of metabolic activity under otherwise identical conditions. Assessment of treatment geometry—specifically the radius of circular motion of the plasma jet—significantly affects metabolic activity, even at identical exposure times. To standardize conditions across devices, we further investigated an indirect treatment approach using a metal grid to generate plasma-conditioned PBS. However, we found a non-linear relationship between liquid volume, treatment time, and biological outcome. Moreover, indirect treatment excludes short-lived reactive species and non-chemical plasma components, limiting its biological relevance. Our findings demonstrate that neither direct nor indirect treatment protocols reliably enable cross-device comparisons in vitro. We therefore advocate for transparent, comprehensive reporting of all device and experimental variables, rather than pursuing a single standardized protocol. This enables meaningful data integration and cross-study comparisons, even when protocols differ.

Abstract Radiation dermatitis (RD) is a common side effect of radiation therapy, potentially leading to treatment interruptions. This study evaluated the efficacy of cold atmospheric plasma (CAP) in a mouse model of RD, specifically focusing on treatment initiation at the peak of disease severity. As secondary objective, hyperspectral imaging (HSI) and optical coherence tomography (OCT), were evaluated as objective measures to monitor disease severity. RD was induced via gamma irradiation, with daily CAP treatment initiated at the peak severity. RD severity was monitored using macroscopic scoring and imaging. We found that CAP treatment initiated at the peak of disease did not result in a measurable improved healing under the tested conditions. HSI showed significant differences ( p ≤ 0.05) between healthy and RD skin even beyond macroscopic scoring. OCT differentiated between healthy and diseased skin, however, no significant difference was observed after macroscopic healing. Our findings suggest that CAP therapy initiated at peak RD does not accelerate recovery. However, this result does not preclude potential efficacy of CAP when administered prophylactically or at the early onset of skin injury. The study underscores the critical importance of treatment timing and highlights HSI and OCT as potentially valuable non-invasive tools for objective RD assessment.

Reactive oxygen and nitrogen species (RONS) generated by medical gas plasmas are considered major mediators of plasma-induced biological effects. This includes the atmospheric pressure argon plasma jet kINPen routinely used in clinical applications. The jet’s biomedical action has been shown to be tailored by modifying its feed gas. However, a systematic comparison of how feed gas composition and humidity shape plasma chemistry remains lacking, which would shift application-specific plasma chemistries from guessing to designing. In this study, we systematically investigated, compared, and statistically related 65 individual feed gas conditions of the kINPen argon plasma jet by increasing O2, N2, and combined O2 + N2 admixtures under dry and humidified conditions. Plasma gas phases were assessed using optical emission spectroscopy and reactive species produced in liquid via hydrogen peroxide, nitrite, and nitrate quantification. O2-containing admixtures generally reduced overall plasma emission and liquid-phase RONS accumulation, whereas N2-containing admixtures preferentially enhanced nitrogen-associated emission features. Water vapor addition via the admixture gas stream acted as an important secondary tuning parameter, exerting the strongest effects under combined O2 + N2 conditions. Multivariate analyses confirmed clear separation of chemistry profiles according to feed gas composition and humidity, while correlation and regression analyses identified several condition-dependent relationships between gas-phase emissions and liquid-phase reaction products. These data provide a comprehensive characterization of kINPen plasma chemistry under controlled feed gas modification and establish a reference framework for tailoring plasma-derived reactive species profiles in future plasma biology and medicine studies.

Chronic wounds place a considerable burden on patients and healthcare systems. Cold plasma technology has become increasingly established for the treatment of people with chronic wounds in recent years. Currently, various cold plasma devices are available. These all generate body-temperature plasmas as well as reactive oxygen and nitrogen species (ROS/RNS). They have not only antimicrobial effects, but also increase dermal blood flow and enhance cell proliferation. Thus, cold plasma-mediated, accelerated wound healing is based on several biological effects that synergistically act within one treatment. Therefore, the AWMF S2k guideline on the rational therapeutic use of cold physical plasma, published in 2022, recommends wound care using cold plasma technology. This article provides an overview of the clinical evidence, potential mechanisms of action, and safety aspects of the therapeutic use of cold plasma technology in chronic wounds.

Cold atmospheric plasma (CAP) enables painless tissue treatment by producing reactive species including excited molecules and charged particles and is of great interest for medical applications. Medical CAP sources work in contact with air at ambient pressure, resulting in the generation of substantial amounts of reactive oxygen and nitrogen radicals. These radicals have a significant influence on cellular biochemistry, are crucial components of the immune system, and play a central role in wound therapy. CAP has a variety of applications, with a particular emphasis on tissue treatment in dermatology. It eradicates microorganisms by preventing biofilm formation so that wounds can be effectively disinfected and treated antiseptically.Using both in vitro and ex vivo methods, a comprehensive preclinical assessment of a novel battery-operated cold plasma handheld device with a reusable, and autoclavable glass cylinder was performed. The objectives were to evaluate the potential impact of single CAP application on radical formation with and without wound dressing, by directly measuring radicals in skin, to investigate the influence of CAP application on antimicrobial activity and cytotoxicity in vitro, and to assess skin tolerance ex vivo.The direct effect of CAP on the formation of radicals in the skin after plasma application at different levels with and without wound dressing was demonstrated quantitatively for the first time using electron paramagnetic resonance spectroscopy. Free radicals were measured in the skin as a function of the duration of CAP treatment. Furthermore, it was found that an alginate or wound plaster dressing does not significantly inhibit radical formation in skin compared to application without a dressing. In vitro and ex vivo data showed no cytotoxic potential with simultaneous efficacy against bacteria strains and no risk of temperature rise, pH change, skin barrier or DNA damage. These results show a high potential for wound healing applications in vivo.

Cold atmospheric pressure plasma (CAP) offers a variety of therapeutic possibilities and induces the formation of reactive chemical species associated with oxidative stress. Mesenchymal stem/stromal cells (MSCs) play a central role in tissue regeneration, partly because of their antioxidant properties and ability to migrate into regenerating areas. During the therapeutic application, MSCs are directly exposed to the reactive species of CAP. Therefore, the investigation of CAP-induced effects on MSCs is essential. In this study, we quantified the amount of ROS due to the CAP activation of the culture medium. In addition, cell number, metabolic activity, stress signals, and migration were analyzed after the treatment of MSCs with a CAP-activated medium. CAP-activated media induced a significant increase in ROS but did not cause cytotoxic effects on MSCs when the treatment was singular and short-term (one day). This single treatment led to increased cell migration, an essential process in wound healing. In parallel, there was an increase in various cell stress proteins, indicating an adaptation to oxidative stress. Repeated treatments with the CAP-activated medium impaired the viability of the MSCs. The results shown here provide information on the influence of treatment frequency and intensity, which could be necessary for the therapeutic application of CAP.

INTRODUCTION:Skin cancer is often fatal, which motivates new therapy avenues. Recent advances in cancer treatment are indicative of the importance of combination treatments in oncology. Previous studies have identified small molecule-based therapies and redox-based technologies, including photodynamic therapy or medical gas plasma, as promising candidates to target skin cancer. OBJECTIVE:We aimed to identify effective combinations of experimental small molecules with cold gas plasma for therapy in dermato-oncology. METHODS:Promising drug candidates were identified after screening an in-house 155-compound library using 3D skin cancer spheroids and high content imaging. Combination effects of selected drugs and cold gas plasma were investigated with respect to oxidative stress, invasion, and viability. Drugs that had combined well with cold gas plasma were further investigated in vascularized tumor organoids in ovo and a xenograft mouse melanoma model in vivo. RESULTS:The two chromone derivatives Sm837 and IS112 enhanced cold gas plasma-induced oxidative stress, including histone 2A.X phosphorylation, and further reduced proliferation and skin cancer cell viability. Combination treatments of tumor organoids grown in ovo confirmed the principal anti-cancer effect of the selected drugs. While one of the two compounds exerted severe toxicity in vivo, the other (Sm837) resulted in a significant synergistic anti-tumor toxicity at good tolerability. Principal component analysis of protein phosphorylation profiles confirmed profound combination treatment effects in contrast to the monotherapies. CONCLUSION:We identified a novel compound that, combined with topical cold gas plasma-induced oxidative stress, represents a novel and promising treatment approach to target skin cancer.

The major risk factor for chronic disease is chronological age, and age-related chronic diseases account for the majority of deaths worldwide. Targeting senescent cells that accumulate in disease-related tissues presents a strategy to reduce disease burden and to increase healthspan. The senolytic combination of the tyrosine-kinase inhibitor dasatinib and the flavonol quercetin is frequently used in clinical trials aiming to eliminate senescent cells. Here, our goal was to computationally identify natural senotherapeutic repurposing candidates that may substitute dasatinib based on their similarity in gene expression effects. The natural senolytic piperlongumine (a compound found in long pepper), and the natural senomorphics parthenolide, phloretin and curcumin (found in various edible plants) were identified as potential substitutes of dasatinib. The gene expression changes underlying the repositioning highlight apoptosis-related genes and pathways. The four compounds, and in particular the top-runner piperlongumine, may be combined with quercetin to obtain natural formulas emulating the dasatinib + quercetin formula.

Clinical therapies, including dermatology and oncology, require safe application. In vitro experiments allow only limited conclusions about in vivo effects, while animal studies in, e.g., rodents have ethical constraints at a large scale. Chicken embryos lack pain reception until day 15 postfertilization, making the in ovo model a suitable alternative to in vivo safety assessment. In addition, the hen's egg test on chorioallantoic membrane assay allows irritation potential analysis for topical treatments, but standardized analysis has been limited so far. Medical gas plasma is a topical, routine, approved dermatology treatment. Recent work suggests the potential of this technology in oncology. Its main mode of action is the release of various reactive species simultaneously. Intriguingly, varying plasma feed gas compositions generates customized reactive species profiles previously shown to be optimized for specific applications, such as skin cancer treatment. To support clinical implications, we developed a novel chicken embryo CAM scoring and study scheme and employed the model to analyze 16 different plasma feed gas settings generated by the atmospheric pressure plasmajet kINPen, along with common anticancer drugs (e.g., cisplatin) and physiological mediators (e.g., VEGF). Extensive gas- and liquid-phase plasma reactive species profiling was done and was found to have a surprisingly low correlation with irritation potential parameters. Despite markedly different reactive species patterns, feed gas-modulated kINPen plasma was equally tolerated compared to standard argon plasma. CAM irritation with gas plasmas but not anticancer agents was reversed 48 h after treatment, underlining the only temporary tissue effects of medical gas plasma. Our results indicate a safe therapeutic application of reactive species.

Centromere (CEN) identity is specified epigenetically by specialized nucleosomes containing evolutionarily conserved CEN-specific histone H3 variant CENP-A (Cse4 in Saccharomyces cerevisiae, CENP-A in humans), which is essential for faithful chromosome segregation. However, the epigenetic mechanisms that regulate Cse4 function have not been fully defined. In this study, we show that cell cycle-dependent methylation of Cse4-R37 regulates kinetochore function and high-fidelity chromosome segregation. We generated a custom antibody that specifically recognizes methylated Cse4-R37 and showed that methylation of Cse4 is cell cycle regulated with maximum levels of methylated Cse4-R37 and its enrichment at the CEN chromatin occur in the mitotic cells. Methyl-mimic cse4-R37F mutant exhibits synthetic lethality with kinetochore mutants, reduced levels of CEN-associated kinetochore proteins and chromosome instability (CIN), suggesting that mimicking the methylation of Cse4-R37 throughout the cell cycle is detrimental to faithful chromosome segregation. Our results showed that SPOUT methyltransferase Upa1 contributes to methylation of Cse4-R37 and overexpression of UPA1 leads to CIN phenotype. In summary, our studies have defined a role for cell cycle-regulated methylation of Cse4 in high-fidelity chromosome segregation and highlight an important role of epigenetic modifications such as methylation of kinetochore proteins in preventing CIN, an important hallmark of human cancers.