Plasma generated RONS in cell culture medium for in vitro studies of eukaryotic cells on Tissue Engineering scaffolds

Abstract

Cold plasmas are continuously developed for biomaterials engineering as well as for therapeutic treatments of cells and tissues. For this last application plasma activated media (PAM) and reactive oxygen and nitrogen species (RONS) gained attention as key players. Here, the use of cold atmospheric pressure (AP) plasma is described, for generating RONS in Dulbecco's Modified Eagles Medium (DMEM); superoxide anion (normalO2−), hydrogen peroxide (H2O2), nitrates (NO3−), and nitrites (NO2−) were detected. PAM was applied to Bone Marrow Stem Cells (BMSC) and SAOS‐2 osteoblasts. Both native and plasma‐modified polymeric scaffolds were used as three dimensional (3D) supports for cell cultures. The cell activity was found dependent on both PAM and cell type. BMSCs grown on plasma‐coated scaffolds tolerated better PAM with respect to those on native scaffolds.

Plasma Treated Water Solutions in Cancer Treatments: The Contrasting Role of RNS

“…We decided to analyze the concentration of NO 2 − and H 2 O 2 used as markers, respectively, of all RNS and ROS species present in the PT-DMEM due to their estimated long life-time also in complex media. The presence of different species like as an example ONOO − and O 2 ●− cannot be in any case excluded [ 37 ]. The results of the chemical composition of PT-DMEM in terms of NO 2 − and H 2 O 2 concentration after N 2 , O 2 ● − and air-DBDs, performed at different treatment times (30–180 s), are reported in Figure 7 .…”

“…For control cells, the original medium was replaced with 2 mL of untreated fresh medium. After 2 h of incubation (time 0 h), PT-DMEM was removed, cells were rinsed once with untreated DMEM, and left growing for 24 and 72 h. After 24 and 72 h cells were stained with a Coomassie Brilliant Blue solution, as described in our previous papers [6,37]: cells were fixed with 4% paraformaldehyde in Phosphate Buffer Saline (PBS) for 20 min, rinsed twice with PBS, then a Coomassie Brilliant Blue (R250, Biorad, Hercules, CA, USA) solution in methanol (45% v/v), acetic acid (10% v/v), and water (45% v/v) was added for 3 min. After rinsing with double distilled water (ddH 2 O), cells were observed using an inverted optic microscope (Nikon Eclipse Ti); at least 7 pictures per time/type/dish were acquired with a Nikon DS Fi2 CCD camera.…”

“…•− cannot be in any case excluded [37] H2O2 (i.e., marker of ROS species) in PT-DMEM increases with the percentage of O2 in the feed, from N2 (where the source of oxygen in the feed comes from water evaporated from the liquid) to pure O2. The NO2 − (i.e., marker of RNS species) concentration in the liquid, instead, is higher when both O2 and N2 are fed in the plasma, as in the case of airtreated DMEM, and decreases in liquids treated with N2-DBD.…”

“…The lack of reliable detection methods of RONS in complex matrices like PTWS, is currently the biggest limitation. Due to the complexity of the study, most of biological effects were reported for PTWS produced by plasma treatment of cell culture media (i.e., liquid media containing carbohydrates, vitamins, growth factors, amino acids, and inorganic salts) to be used in vitro [11,[34][35][36][37] while, for sake of simplicity, most of the chemical studies investigating RNS formation in PTWS have been performed in simple model liquids like water or similar [38]. With specific regard to • NO, however, chemical routes of formation in PTWS seems to be strongly affected not only by the environment where the plasma is ignited, but also by the chemical composition of the liquid to be treated.…”

The emerging role of cold atmospheric plasma in glioblastoma therapy

“…[ 65,66 ] As illustrated in Figure 1, the CAP‐originated RNOS play an important role in the anticancer mechanism of CAP in vitro. [ 67 ]…”