Plasma-treated Medium Research Articles

Effects of cold atmospheric plasma‐treated medium on HaCaT and HUVEC cells in vitro

AbstractCold atmospheric plasma (CAP) is an emerging technology that can generate various reactive oxygen and nitrogen species (RONS) at room temperature and shows promising applications for skin wound healing. The effects of plasma‐treated medium (PTM) promoting cell proliferation have been verified, but the biological mechanisms involved are not well known. This study aims to assess the proliferation effect and mechanism induced by PTM on human keratinocyte cells (HaCaT) and human umbilical vein endothelial cells (HUVEC) in vitro. The results showed that the concentrations of H2O2 and NO2− inside PTM increased in a time‐dependent manner as the atmospheric pressure plasma jet (APPJ) treatment time was prolonged. The biological outcomes were determined by cell counting kit, wound healing assay, flow cytometry, enzyme‐linked immunosorbent assay (ELISA), and western blot analysis. The cell viability and motility assays indicated that appropriate plasma conditions of short treatment time (15s‐, 30s‐ and 45s‐PTM) could promote cell proliferation, while long treatment time would inhibit cell proliferation (60s‐PTM). With an appropriate time of PTM treatment, the secretion of vascular endothelial growth factor‐α (VEGF‐α) and transforming growth factor alpha (TGF‐α) was promoted and the extracellular signal‐regulated kinase/serine‐threonine protein kinase pathways were activated, which induced HaCaT and HUVEC cell proliferation eventually. These behaviors of cells were mainly related to the enhancement of intracellular reactive oxygen species levels. These findings established a theoretical foundation for potential clinical applications of PTM in wound healing.

Increased Expression of ITGB 3 in CLL Patient leukemia Cells by Exposure to Cold Physical Plasma and Plasma-treated Medium.

Chronic lymphocytic leukemia (CLL) is the most prevalent hematological cancer, with various medical interventions. In the recent decade, cold physical plasma has become an interesting agent for future cancer therapy. The goal of this study was to see whether cold physical plasma or cold physical plasma-treated liquid (PTL) affected integrin beta 3 (ITGB3) expression, which is hypothesized to mediate an interaction between cancer stem cells and the bone marrow microenvironment, in CLL patients' blood cells. The metabolic activity, cell death pattern, lipid oxidation and ITGB3 gene expression of these treatments was evaluated. Both direct cold physical plasma and PTL exposure enhanced lipid peroxidation in cells of CLL patients, but to a lesser extent in healthy participants. Furthermore, following 48h of cold physical plasma or PTL exposure, the metabolic activity of leukocytes was preferentially reduced in CLL patient leukocytes. In addition, cold physical plasma and PTL treatment elevated ITGB3 mRNA expression in CLL patients' leukocytes compared to untreated and healthy controls. Collectively, our study suggests selective effects of direct cold physical plasma and PTL exposure on blood leukocytes from leukemia patients, but further and more detailed studies are needed to provide additional rationales for such treatment options as future therapy.

Cold plasma-treated medium preferentially eliminates doxorubicin-resistant osteosarcoma cells

Osteosarcoma (OS) is an aggressive bone cancer with poor prognosis, largely due to the limited effectiveness of current treatments such as doxorubicin (DX). Developing ways to overcome DX resistance is a significant clinical challenge. Here, we used two DX-resistant models to study the potential of Cold Plasma Treated Medium (PTM) to prevent DX resistance in OS. During the acquisition of the resistant phenotype upon long-term DX exposure, OS resistant cells became less proliferative, overexpressed the drug resistance-related efflux pump MDR1 and displayed a concomitant loss of SOD2 or GPX1. According to the reduced expression of these antioxidant enzymes, PTM treatment produced higher levels of oxidative express and was more effective in eradicating DX-resistant cells. Moreover, PTM reduced the expression of MDR1, thus sensitizing resistant cells to DX. These findings uncover new vulnerabilities of DX-resistant cells related with their inability to cope with excessive oxidative stress and their dependence on MDR1 that can be exploited using PTM-based treatments to provide new therapeutic approaches for the management of drug resistance in OS.

Effect of Plasma On-Time with a Fixed Duty Ratio on Reactive Species in Plasma-Treated Medium and Its Significance in Biological Applications

Optimizing the therapeutic range of nonthermal atmospheric pressure plasma (NTAPP) for biomedical applications is an active research topic. For the first time, we examined the effect of plasma on-times in this study while keeping the duty ratio and treatment time fixed. We have evaluated the electrical, optical, and soft jet properties for two different duty ratios of 10% and 36%, using the plasma on-times of 25, 50, 75, and 100 ms. Furthermore, the influence of plasma on-time on reactive oxygen and nitrogen species (ROS/RNS) levels in plasma treated medium (PTM) was also investigated. Following treatment, the characteristics of (DMEM media) and PTM (pH, EC, and ORP) were also examined. While EC and ORP rose by raising plasma on-time, pH remained unchanged. Finally, the PTM was used to observe the cell viability and ATP levels in U87-MG brain cancer cells. We found it interesting that, by increasing the plasma on-time, the levels of ROS/RNS dramatically increased in PTM and significantly affected the viability and ATP levels of the U87-MG cell line. The results of this study provide a significant indication of advancement by introducing the optimization of plasma on-time to increase the efficacy of the soft plasma jet for biomedical applications.

Knockdown of Peroxiredoxin V increased the cytotoxicity of non-thermal plasma-treated culture medium to A549 cells.

Administration of non-thermal plasma therapy via the use of plasma-activated medium (PAM) might be a novel strategy for cancer treatment, as it induces apoptosis by increasing reactive oxygen species (ROS) levels. Peroxiredoxin V (PRDX5) scavenges ROS and reactive nitrogen species and is known to regulate several physiological and pathological reactions. However, its role in lung cancer cells exposed to PAM is unknown. Here, we investigated the effect of PRDX5 in PAM-treated A549 lung cancer cells and determined the mechanism underlying its cytotoxicity. Cell culture medium was treated with low temperature plasma at 16.4 kV for 0, 60, 120, or 180 s to develop PAM. PRDX5 was knocked down in A549 cells via transfection with short hairpin RNA targeting PRDX5. Colony formation and wound healing assays, flow cytometry, fluorescence microscopy, and western blotting were performed to detect the effect of PRDX5 knockdown on PAM-treated A549 cells. PAM showed higher cytotoxicity in lung cancer cells than in control cells, downregulated the mitogen-activated protein kinase signaling pathway, and induced apoptosis. PRDX5 knockdown significantly inhibited cell colony formation and migration, increased ROS accumulation, and reduced mitochondrial membrane potential in lung cancer cells. Hence, PRDX5 knockdown combined with PAM treatment represents an effective option for lung cancer treatment.

The Effects of Plasma-Activated Saline and Medium on Colorectal Cancer in Rat Models and the Human Colon Adenocarcinoma Cell Line COLO 205

Colorectal cancer affects a large number of people. There is an urgent need to develop novel treatments for this disease. In recent years, research on medical applications of atmospheric low temperature plasma (ALTP) has been actively conducted, and cancer treatment is one of the targets. In this study, we focused on colorectal cancer and evaluated plasma as a new treatment method. In vivo and in vitro experiments were conducted using plasma-treated saline (PTS) and plasma-treated medium (PTM) prepared by submerged bubbling treatment using ALTP. In vivo experiments using a rat model of colorectal cancer revealed that PTS administration to the colon slowed tumor progression based on endoscopic and histopathological observations. To investigate the cause of the inhibition of tumor progression, we evaluated the impact of ALTP on colon cancer cells. As a result of culturing colorectal cancer cells with PTM, cell proliferation was inhibited. Also, cell death was induced by cell swelling. qPCR revealed that PTMs induced cell death of cancer cells through signaling of tumor necrosis factor α (TNF-α), an inflammatory cytokine. Therefore, the inhibition of cancer progression by PTS in the rat model occurs by inducing cell death through the dissolution of ALTP components in the liquid.

Absorption of FD-150 into Intestinal Cells by Microplasma

Introduction of molecules into the cells is part of gene transfection or cell-mediated drug delivery. The molecules can effectively decrease side effects of the drug and increase their targeting. Air microplasma discharge was used to treat rat intestinal cells to induce absorption of fluorescein isothiocyanate-dextran with a molecular weight of 150 kDa (FD-150). Absorption of FD-150 was confirmed by observation using fluorescence microscope. Efficiency of absorbance was estimated by a plate reader, and the viability of the cells was measured by a cell counter. The viability of the cells was proportional to the decrease in treatment time and the increase in treatment distance from the microplasma electrode. The absorption of FD-150 was found to be proportional to the increase in time and distance. No improvement in absorption or viability was observed during the incubation time. Cell treatment by the plasma-treated medium showed a lower absorption of FD-150 than direct treatment but the same cell viability. Concentration of ozone in the gas phase and OH radical in the liquid phase were observed.

Analysing Mouse Skin Cell Behaviour under a Non-Thermal kHz Plasma Jet

Plasma jets are extensively used in biomedical applications, particularly for exploring cell viability behaviour. However, many experimental parameters influence the results, including jet characteristics, secondary liquid chemistry and protocols used, slowing research progress. A specific interest of the presented research was skin cell behaviour under a non-thermal kHz plasma jet—a so-called cold plasma jet—as a topical skin treatment. Our research was focused on in vitro mouse skin cell direct plasma treatment with argon as an operating gas. The research was complemented with detailed gas-phase diagnostics and liquid-phase chemical analysis of the plasma and plasma-treated medium, respectively. The obtained results showed that direct plasma jet treatment was very destructive, leading to low cell viability. Even with short treatment times (from 35 s to 60 s), apoptosis was observed for most L929 murine fibroblasts under approximately the same conditions. This behaviour was attributed to plasma species generated from direct treatment and the types of cell lines used. Importantly, the research exposed important points that should be taken under consideration for all further research in this field: the urgent need to upgrade and standardise existing plasma treatment protocols of cell lines; to monitor gas and liquid chemistries and to standardise plasma discharge parameters.

Indirect Cold Atmospheric Pressure Plasma treatment on Melanoma and Fibroblast Cells

Cold atmosphere plasma has been shown as a promising technology for certain cancer treatments. In this paper, we report indirect plasma treatment using CAP discharged in cell culture medium and study the effect of identical plasma stimulated culture medium on melanoma cancer cells and fibroblast cells cultured in vitro. The results of MTT assay, migration assay, ROS detection, and alizarin red assay show that plasma-treated medium can have a strong negative effect on melanoma cancer cells compared with the control group. However, the plasma-treated medium has a less cytotoxic effect on fibroblast cells than that on melanoma cancer cells at the same treatment. This result is attributed to the production of reactive oxygen species in the plasma-treated medium to induce apoptosis and inhibit melanoma cell proliferation and further cell metastasis. According to the results, this study shows the potential of CAP plasma treatment for anti-cancer therapy.

Analysis of Hydroxyl Radical and Hydrogen Peroxide Generated in Helium-Based Atmospheric-Pressure Plasma Jet and in Different Solutions Treated by Plasma for Bioapplications

This study systematically analyzed reactive species generated with self-built helium-based low-temperature atmospheric-pressure plasma jet (He-APPJ); we measured the hydroxyl radical (·OH) and hydrogen peroxide (H2O2) above surfaces of plasma–treating solutions and in plasma-treated medium (PTM) of different solutions, including deionized water, phosphate buffered saline, and Dulbecco’s modified Eagle medium. The effects of adding O2 into He working gas on ·OH and H2O2 generation were discussed. The MCF7 cell responses to plasma treatment were also observed. The ·OH emission (309 nm) in He-APPJ and above surfaces of plasma–treating solutions were analyzed through optical emission spectroscopy, and concentrations of ·OH and H2O2 in different solutions were measured using terephthalic acid and Amplex Red, respectively. In our plasma system, it was observed that higher conductivity of solutions may cause stronger ·OH emission above solution. The ingredients of the solution may affect the concentrations of ·OH and H2O2. Addition of 0.1% O2 generates strongest ·OH emission above surfaces of plasma–treating solution with our He-APPJ, and thus, the highest H2O2 concentration in PTM. The MCF7 cell viability is influenced by the H2O2 concentration in PTM. The results of this study can facilitate further study of plasma effects on PTM and cell culture.

Quantifying the concentration and penetration depth of long-lived RONS in plasma-activated water by UV absorption spectroscopy

Reactive oxygen and reactive nitrogen species (RONS) are believed to play a key role in biomedical applications, which means that RONS must reach the target tissue to produce a therapeutic effect. Existing methods (electron spin spectrometry and microplate reading) to determine the RONS concentration are not suitable for experimental real-time measurements because they require adding an indicating reagent to the plasma-treated medium, which may alter the chemical composition of the medium. In this paper, we propose a method to measure the long-lived RONS concentration in plasma-activated water (PAW) by using UV absorption spectroscopy. Based on an analysis and fit of the absorption spectra of standard solutions (H2O2, NaNO2, and NaNO3), we propose a detailed fitting procedure that allows us to calculate the concentrations of simplex H2O2, NO2−, and NO3−. The results show that the pH and the cross reactivity between RONS in PAW correlate strongly with the absorption spectra. To confirm the accuracy of the calculations, we also use a microplate reader and add chemical reagents to measure the concentrations of H2O2, NO2−, and NO3−. The results show that the concentrations calculated by the proposed fitting method are relatively accurate and that the error range is acceptable. Additionally, the time-dependent diffusion of RONS in PAW is measured and analyzed at different depths in the PAW. This fitting approach constitutes a nonintrusive approach to measure RONS at different depths in PAW.

Hmox1 Upregulation Is a Mutual Marker in Human Tumor Cells Exposed to Physical Plasma-Derived Oxidants.

Increasing numbers of cancer deaths worldwide demand for new treatment avenues. Cold physical plasma is a partially ionized gas expelling a variety of reactive oxygen and nitrogen species, which can be harnesses therapeutically. Plasmas and plasma-treated liquids have antitumor properties in vitro and in vivo. Yet, global response signatures to plasma treatment have not yet been identified. To this end, we screened eight human cancer cell lines to investigate effects of low-dose, tumor-static plasma-treated medium (PTM) on cellular activity, immune-modulatory properties, and transcriptional levels of 22 redox-related genes. With PTM, a moderate reduction of metabolic activity and modest modulation of chemokine/cytokine pattern and markers of immunogenic cell death was observed. Strikingly, the Nuclear factor (erythroid-derived 2)-like 2 (nrf2) target heme oxygenase 1 (hmox1) was upregulated in all cell lines 4 h post PTM-treatment. nrf2 was not changed, but its baseline expression inversely and significantly correlated with hmox1 expression after exposure to PTM. Besides awarding hmox1 a central role with plasma-derived oxidants, we present a transcriptional redox map of 22 targets and chemokine/cytokine secretion map of 13 targets across eight different human tumor cell lines of four tumor entities at baseline activity that are useful for future studies in this field.

A myeloid and lymphoid infiltrate in murine pancreatic tumors exposed to plasma-treated medium

PurposeMetastatic pancreatic cancer often is fatal in patients. Palliation can include disseminating large amounts of chemotherapeutic liquid in the peritoneal cavity to slow tumor growth. We have previously demonstrated that repeated application of plasma-treated medium performed well in decreasing peritoneal tumor burden in mice and prolonging animal survival. We here extend on this study by detailed immune-related analysis of cryo-conserved tumor nodes of mice that had received either cell culture medium alone or plasma-treated cell culture medium. MethodsAnimals of the treatment group had significantly fewer lesions, which were characterized by an in-creased influx of macrophages. The staining intensity of CD206, a murine M2 macrophage marker associated with tumor promotion, was decreased in tissue sections, while iNOS (M1 marker associated with inflammatory macrophages) was not changed. In the infiltrate, other myeloid cells such as neutrophils and dendritic cells were in tendency increased and decreased, respectively. Further, we observed a significant increase in T cells and calreticulin staining, suggesting an involvement of immunogenic cancer cell death in plasma-treated medium therapy of pancreatic tumor lesions. ConclusionIn summary, exposure to plasma-treated medium not only decelerates tumor growth but also serves as immunomodulatory agent with a possible relevance for therapeutic outcome.

Plasma-treated medium tunes the inflammatory profile in murine bone marrow-derived macrophages

PurposeMacrophages are essential drivers of tumor rejection as well as tumor promotion. Especially tumor-associated macrophages (TAM) phenotypically resemble tumor-supporting alternatively activated macrophages (M2). Targeting their phenotype has long been a matter of preclinical research in oncology. Cold physical plasma and plasma-treated medium has recently been recognized as a new possible interventional strategy in tumor treatment. Whereas several studies underlined this proof-of-concept in animal studies, it is not clear how plasma affects the phenotype of macrophages. MethodsWe differentiated macrophage from murine bone marrow-derived cells, and exposed them to plasma-treated cell culture medium. This led to a more pronounced NOS2 expression in several macrophage subtypes, a marker typically associated with a rather pro-inflammatory, antitumor phenotype. When stimulated with supernatants of pancreatic cancer cells, these macrophages released significantly increased amounts of immune-stimulatory molecules in response to plasma-treated medium. This included TNFα, IL6, IL12, CCL4, and CXCL9, whereas MCP1 and CXCL1 were significantly decreased. Interestingly, baseline expression levels as well as response to plasma-treated medium were largely opposite to macrophages stimulated with tumor cell supernatants. ConclusionThese results call for a more differentiated view on macrophage polarization, and emphasize the immune-modulatory role that plasma-treated medium may exert in the tumor settings.

Mitochondrial Complex IV Inhibition and Exogenous Oxidants Potently Synergize in Melanoma Cell Death Induction

Due to its functional duality in toxicity as well as signaling, research on therapeutic manipulation of reactive oxygen species (ROS) in cancer has been futile. Targeting melanoma cell death with excess amounts ROS, however, is difficult as these cells are highly resistant towards oxidative stress, creating a need for alternative and combinational approaches. Tumor metabolism mainly relies on aerobic glycolysis. We investigated a potential synergism of mitochondrial complex IV inhibition and exogenous-derived oxidants with both single treatment modalities being essentially non-toxic in melanoma cells. In this study, we investigated murine (metastatic B16F10 and non-metastatic B16F0) as well as human (SK-Mel 28) melanoma cells, and compared them with murine (primary fibroblasts) and human (HaCaT keratinocytes) non-cancer cells. Mitochondrial complex IV inhibition was realized using sodium azide (NaN3) and potassium cyanide (KCN) and pharmacological inhibitor ADDA5. Exogenous oxidants were generated using cell culture medium that has been treated with the cold physical plasma effluent of an atmospheric pressure argon plasma jet (kINPen). This plasma-treated medium was subsequently applied to different cells. Metabolic activity was assessed by fluorescence quantification of resazurin-to-resorufin transformation. ATP levels were monitored by a luminescence assay. Total cell counts, intracellular ROS levels (DCF), mitochondria membrane potential (TMRE), cell membrane integrity (sytox green) and cellular morphology were quantitatively assessed using live cell and high throughput confocal imaging (Operatta; Perkin Elmer). For tumor cells, also 3D spheroid models were investigated for viability by sytox green staining. ADDA5, NaN3 and KCN treatment alone failed to induce a significant alteration in metabolic activity in cancer cells. We then exposed the cells pretreated with NaN3 (100-500 µM) or KCN (250-500 µM) to plasma-treated medium. There was a significant inhibition of metabolic activity and rapid induction of cell death 6 h following treatment in melanoma cells compared to cell receiving plasma-treated medium alone. Increase in cellular superoxide levels and decrease in mitochondrial membrane potential and ATP levels was observed preceding cell death. However, no caspase 3 cleavage was observed upon immunoblotting. siRNA mediated knockdown of COX4a led to increased sensitivity of tumor cells towards plasma-treated medium. Furthermore, we validated our findings using a pharmacological inhibitor of complex IV ADDA5 in a tumor spheroid model and demonstrated that complex IV inhibition supplemented with plasma-treated medium, triggered significant cell death via caspase independent mechanism in melanoma cells.

A study of the effect on human mesenchymal stem cells of an atmospheric pressure plasma source driven by different voltage waveforms

The effect of an atmospheric pressure non-equilibrium plasma on human mesenchymal stem cells was investigated. A dielectric barrier discharge non-equilibrium plasma source driven by two different high-voltage pulsed generators was used and cell survival, senescence, proliferation, and differentiation were evaluated. Cells deprived of the culture medium and treated with nanosecond pulsed plasma showed a higher mortality rate, while higher survival and retention of proliferation were observed in cells treated with microsecond pulsed plasma in the presence of the culture medium. While a few treated cells showed the hallmarks of senescence, unexpected delayed apoptosis ensued in cells exposed to plasma-treated medium. The plasma treatment did not change the expression of OCT4, a marker of mesenchymal stem cell differentiation.

Effects of atmospheric pressure cold plasma on human hepatocarcinoma cell and its 5-fluorouracil resistant cell line

Atmospheric pressure cold plasma showed selective killing efficiency on cancer cells in vitro and in vivo, which makes plasma a potential option for cancer therapy. However, the plasma effects on chemotherapeutic drugs-resistant cells are rarely to be found. In this paper, the effects of plasma on human hepatocellular carcinoma Bel7402 cells and 5-fluorouracil (5-FU) resistant Bel7402/5FU cells were intensively investigated. The results showed that plasma induced superior toxicity to Bel7402 cells compared with Bel7402/5FU cells. Incubation with plasma-treated medium for 20 s induced more than 85% death rate in Bel7402 cells, while the same death ratio was achieved when Bel7402/5FU cells were treated for as long as 300 s. The hydrogen peroxide in the medium played a leading role in the cytotoxicity effects. Further studies implicated that when the treatment time was shorter than 60 s, the depolarization of mitochondrial membrane potential and apoptosis occurred through the intracellular reactive oxygen species accumulation in Bel7402 cells. Molecular analysis showed an increase in the transcription factor activity for AP-1, NF-кB, and p53 in Bel7402 cells. No obvious damage could be detected in plasma-treated Bel7402/5FU cells due to the strong intracellular reactive oxygen stress scavenger system.

Cell migration and adhesion of a human melanoma cell line is decreased by cold plasma treatment

Treatment of tumor metastasis continues to be of major importance in the field of cancer medicine. The biological responses of the human melanoma cancer cell line SK-Mel-147 after exposure to cold plasma-treated medium were investigated. Plasma reduced tumor cell motility and colony formation while cell metabolism, apoptosis, and cell cycle progression were less affected. Plasma treatment was associated with an inhibition of migration and disorganization of the actin cytoskeleton which were mediated through multiple signaling pathways, as transcriptome-wide gene analysis suggested. These results provide evidence that cold plasma may be beneficial in melanoma treatment.

Persistent effectivity of gas plasma-treated, long time-stored liquid on epithelial cell adhesion capacity and membrane morphology.

Research in plasma medicine includes a major interest in understanding gas plasma-cell interactions. The immediate application of gas plasma in vitro inhibits cell attachment, vitality and cell-cell contacts via the liquid. Interestingly, in our novel experiments described here we found that the liquid-mediated plasma effect is long-lasting after storage up to seven days; i. e. the liquid preserves the characteristics once induced by the argon plasma. Therefore, the complete Dulbecco's Modified Eagle cell culture medium was argon plasma-treated (atmospheric pressure, kINPen09) for 60 s, stored for several days (1, 4 and 7 d) at 37°C and added to a confluent mouse hepatocyte epithelial cell (mHepR1) monolayer. Impaired tight junction architecture as well as shortened microvilli on the cell membrane could be observed, which was accompanied by the loss of cell adhesion capacity. Online-monitoring of vital cells revealed a reduced cell respiration. Our first time-dependent analysis of plasma-treated medium revealed that temperature, hydrogen peroxide production, pH and oxygen content can be excluded as initiators of cell physiological and morphological changes. The here observed persisting biological effects in plasma-treated liquids could open new medical applications in dentistry and orthopaedics.

Nonthermal Dielectric Barrier Discharge (DBD) Plasma Suppresses Herpes Simplex Virus Type 1 (HSV-1) Replication in Corneal Epithelium.

Herpes keratitis (HK) is the leading cause of cornea-derived and infection-associated blindness in the developed world. Despite the availability of effective antivirals, some patients develop refractory disease, drug-resistant infection, and topical toxicity. A nonpharmaceutical treatment modality may offer a unique advantage in the management of such cases. This study investigated the antiviral effect of nonthermal dielectric barrier discharge (DBD) plasma, a partially ionized gas that can be applied to organic substances to produce various biological effects. Human corneal epithelial cells and explanted corneas were infected with herpes simplex virus type 1 (HSV-1) and exposed to culture medium treated with nonthermal DBD plasma. The extent of infection was measured by plaque assay, quantitative PCR, and Western blot. Corneal toxicity assessment was performed with fluorescein staining, histologic examination, and 8-OHdG detection. Application of DBD plasma-treated medium to human corneal epithelial cells and explanted corneas produced a dose-dependent reduction of the cytopathic effect, viral genome replication, and the overall production of infectious viral progeny. Toxicity studies showed lack of detrimental effects in explanted human corneas. Nonthermal DBD plasma substantially suppresses corneal HSV-1 infection in vitro and ex vivo without causing pronounced toxicity. Nonthermal plasma is a versatile tool that holds great biomedical potential for ophthalmology, where it is being investigated for wound healing and sterilization and is already in use for ocular microsurgery. The anti-HSV-1 activity of DBD plasma demonstrated here could be directly translated to the clinic for use against drug-resistant herpes keratitis.