Non essential amino acids (neaa)

Normal human dermal fibroblasts (NHDF) were isolated from skin following plastic surgery interventions after approval by the local ethics committee of the University Hospital in Pilsen, E. Benese 13, 305 99 Pilsen, Czech Republic, decision of November 5th, 2015. The guidelines in the Declaration of Helsinki were followed. All donors gave written informed consent before intervention. The samples were washed by Hank’s balanced salt solution (HBSS) (Merck KGaA, Darmstadt, Germany) containing penicillin (100 U/ml)/streptomycin (0.1 mg/ml) (Biochrom, Cambridge, UK) and gentamicin (50 μg/ml) (Biochrom). The samples were cut and digested overnight at 37 °C in HBSS containing collagenase type I (100 U/ml, Merck KGaA). On the following day, the suspension was shaken intensively and filtered through 100 µm nylon cell strainer (Falcon™, Thermo Fisher Scientific, Waltham, Massachusetts). The cell suspension was then transferred to a cultivation flask (TPP) containing low glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (Merck KGaA), 10% heat-inactivated fetal bovine serum (FBS) (Merck KGaA), penicillin (100 U/ml)/streptomycin (0.1 mg/ml) (Biochrom), 0.5% L-glutamin (Biosera, Nuaille, France) and 1.0% non-essential amino acids (Biosera). The NHDF were cultured at 37 °C, 5% CO₂ up to 80% confluence and then passaged. Only those NHDF from the 2th–5th passages were used in the experiments.

Twenty NBL cell lines derived in our laboratory from NBL tumor samples as described above and two reference NBL cell lines were included in this study. Both reference cell lines were purchased from the European Collection of Cell Cultures (ECACC): SH-SY5Y (ECACC cat. no. 94030304) and SK-N-BE(2) (ECACC cat. no. 95011815; MYCN amp.) and were reported as RA-sensitive [19 (link)]. The cells were grown in a 1:1 mixture of Dulbecco´s modified Eagle´s medium (DMEM) and Ham´s F 12 medium supplemented with 20% fetal calf serum, 2 mM glutamine, 100 IU/mL penicillin and 100 μg/mL streptomycin (all purchased from GE Healthcare Europe GmbH, Freiburg, Germany) and 1% nonessential amino acids (purchased from Biosera, Nuaille, France). The cell lines were maintained under standard conditions at 37°C in a humidified atmosphere containing 5% CO2 and subcultured 1-2 times weekly.

Human neuroblastoma SH-SY5Y cells, hepatoblastoma HepG2 cells, murine macrophage-like RAW 264.7 cells, and fibroblast-like MRC5 cells from normal lung tissue were all purchased from the American Type Culture Collection (ATCC, VA, United States). HepG2, SH-SY5Y, and MRC5 were cultured in Minimum Essential Medium (MEM; Biosera, France) with 5 mmol/L glucose, and supplemented with both 2 mmol/L glutamine, 10% fetal bovine serum (FBS; Biosera), as well as with 1% non-essential amino acids (Biosera) in a normoxic CO₂ chamber at 37°C in a humidified atmosphere. RAW 264.7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM with 4,500 mg/L glucose, L-glutamine, and sodium bicarbonate) with 10% FBS.
The medium was replaced 24–48 h prior, starting the experiments with fresh medium containing LR and BR in a final concentration of 5, 25, or 50 μmol/L (control samples were treated by medium containing an adequate volume of LR/BR solvents; final concentration of DMSO = 0.5% v/v, concentration of BSA in FBS = 2.5 g/L (Soutar et al., 2019 (link))). Thus, the respective concentrations of Bf (Bilirubin free, unbound, biologically active fraction of bilirubin) corresponded to non-toxic, borderline toxic and toxic concentrations, respectively (Roca et al., 2006 (link); Zelenka et al., 2012 (link)). On the day of the experiment, the control cell culture reached a confluence of 80–90%.

The human colon adenocarcinoma cell lines Caco-2 (American Type Culture Collection ECACC 86010202) were cultured in Eagle’s minimal essential medium (Lonza, Verviers, Belgium) supplemented with 1% (v/v) of non-essential amino acids (Biosera, Boussens, France), 1% (v/v) of pyruvate (Lonza) and 20% (v/v) of fetal bovine serum (FBS; Biowest, Nuaillé, France) and were incubated at 37 °C and at 5% CO₂–95% air atmosphere. The growth medium was replaced with fresh every second day.