Cb210

Lymphocytes are the cell type most often used when investigating DNA damage [36 (link)]. Especially the human TK6 lymphocyte cell line has been widely utilized in genotoxicity studies [37 (link), 38 (link)]. For this reason, we used TK6 (ATCC CRL-8015) cells, a p53-competent, human lymphoblast cell line. Cells were cultured in Roswell Park Memorial Medium without phenol red (RPMI1640; PanBioTech) supplemented with 10% fetal bovine serum, 2% glutamine, and 1% penicillin/streptomycin (all Sigma). All incubations were performed in cell culture conditions (CB210; Binder) at 37°C, 95% humidity, and 5% carbon dioxide. As ROS scavengers, catalase (cat; 20 μg/ml), glutathione (GSH; 1 mM), or superoxide dismutase (SOD; 100 U/ml) was used (all Sigma). As enzyme or signaling inhibitors, Z-VAD-FMK (R&D Biosciences), SB202190 (Sigma), KU55933 (SelleckChem), Ly294002 (Cell Signaling Technologies), wortmannin (InvivoGen), or SP600125 (Santa Cruz Biotechnology) was used at different concentrations and incubated with cells for 1 h prior ROS or UV treatment. Final concentrations for a selected choice of inhibitors were 1 μM for KU55933, 1 μM for SB202190, and 25 μM for Z-VAD-FMK.

The experiments with the use of human cell culture in vitro were carried out following the human experiment issues of the Code of Ethics of the World Medical Association (Declaration of Helsinki, 19 October 2013). In all cases, donors of ADSCs were the informed voluntary. Adipose-derived Stem Cells (ADSCs) were isolated from the lipoaspirate by enzymatic digestion in 0.1% collagenase IA and 0.1% pronase with 2% fetal bovine serum (FBS). This cell suspension was then transferred to 25 cm² cell culture flask (SPL, Gyeonggi-do, Korea) and cultured in the following control growth medium: modified MEM-α with 10% FBS, 2.0 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 1.0 ng/mL bFGF-2. (All media and reagents were obtained from Sigma-Aldrich, St. Louis, MO, USA.) The cells were cultured in multi-gas incubator CB210 (Binder, Tuttlingen, Germany) at +37 °C under saturated humidity, 5% CO₂ and 5% O₂. For the cytotoxicity assessment, the cells were seeded on scaffolds at a density of 2 × 10⁵ cells per 1.0 cm³ of materials. After 48 h of incubation, samples were stained with PI (propidium iodide) and FDA (fluorescein diacetate). The number of dead and living ADSCs in different groups was counted, using fluorescence microscopy (FITC and Texas Red filters; Carl Zeiss, Jena, Germany) and ZEN 2012 software [22 (link)].

Detailed description of the methods was presented in previous study [22 (link)]. Human cancer cell lines A549 (lung carcinoma), HT29 (colon adenocarcinoma), and U87 (glioblastoma) were cultured in DMEM prepared from the powder by dilution in the DDW or Milli-Q water with natural isotopic content and sterilized by filtering. The media are further referred to as DDW medium and control medium. The culture media were additionally supplemented with 10% FBS and 2 mM glutamine (all from Sigma, USA). The cells were cultured in CO₂ incubators CB210 (Binder, Germany) at +37°C in the atmosphere of absolute humidity and 5% CO₂.
A549, HT29, and U87 cell lines were seeded in 24-well or 6-well plates at a density to reach 90% confluence in 24 hours. Afterwards, the cells were cultured in serum-free medium (to inhibit cell proliferation). Then, the cells were washed three times and the culture medium was completely changed for the experimental medium for each group.
To determine the effect of deuterium on the biological properties of A549, HT29, and U87 cell lines, three cell lines at P2 were assessed, after 6 days of culturing in experimental media.
In the experiment with cancer human cell lines in vitro (A549 (lung carcinoma), HT29 (colon adenocarcinoma), and U87 (glioblastoma)), material from American Type Culture Collection (ATCC) was used.