Humidified incubator

Adipose-derived mesenchymal stem cells (ADMSC, ATCC-PCS-500-011) at 5 × 10³ were seeded onto the scaffolds in the 96-well plates, as in the standard cell seeding procedure, after 4 h ultraviolet (UV) light sterilization. At the same time, monolayer cell cultures were incubated with the same number of cells in 150 μL as a control. The cell-scaffold constructs and monolayer cultures were incubated at 37 °C, 5% CO₂ for 1 and 3 days in a humidified incubator (NuAire). The toxic effect of scaffolds was checked on days 1 and 3. To investigate cytotoxicity at a given time point, the MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) (Glentham Life Sciences) cytotoxicity assay method was used according to the manufacturer’s protocol. The absorbance value of the cytotoxicity test was measured at 570 nm wavelength (690 nm as Ref. value) in an ELISA reader (Enspire, Perkin Elmer, Waltham, MA, USA). The assay was studied 3 times, and the average of the results was considered the final result.

The human hepatoma cell line (HepG2) was purchased from the American Type Culture Collection (ATCC HB-8065). These cells were cultured in Minimum Essential Medium Eagle (MEME) (ATCC). The murine fibroblast cell line (Balb/c 3T3 clone A31) was purchased from the American Type Culture Collection (ATCC CCL-163), and the cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC). The media were supplemented with 10% BCS (Balb/c 3T3), 10% FBS (HepG2), 1% l-glutamine, and 1% antibiotic solution. The cells were maintained in 75-cm² cell culture flasks (NUNC, Roskilde, Denmark) in a humidified incubator, NuAire (Plymouth, MN, USA) at 37 °C and 5% CO₂. The medium was refreshed every two days and the cells were trypsinized by 0.25% trypsin–0.02% EDTA after reaching 70–80% confluence. Then, cell suspensions at a density of 2 × 10⁵ cells/mL (HepG2) and 5 × 10⁴ cells/mL (Balb/c 3T3) were transferred to 96-well plates (100 μL/well) and incubated for 24 h before the exposure to the studied compounds.

Murine C8-B4 microglia cells (ATCC CRL-2540; monocytes from cerebellum of C57BL/6 mice (Mus musculus) and C8-D1A astrocytes (ATCC CRL-2541; astrocyte type I clone from C57BL/6 mouse cerebellum) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in DMEM-supplemented with 10% FBS and 1% penicillin/streptomycin. The microglia and astrocytes were cultured at 37 °C under 5% CO₂ in a humidified incubator (NuAire, Plymouth, MN, USA) and sub-cultivated by trypsination once/twice per week in a 1:5/1:10 split ratio, respectively. They were allowed to grow as monolayers in 75 cm² cell culture flasks with filter screwcaps (Techno Plastic Products, Trasadingen, Switzerland) until 80–100% confluence was reached. The microglia and astrocytes were plated at 90,000/cm² and 45,000/cm², respectively, 24 h prior to the test, which resulted in approximately 90% confluence at the day of exposure as observed by light microscopy (Leica DMIL, Solms, Germany). The cell culture medium was refreshed before performing the exposure test.

The human hepatoma cell line (HepG2) was purchased from the American Type Culture Collection (ATCC HB-8065). These cells were cultured in Minimum Essential Medium Eagle (MEME) (ATCC). The murine fibroblast cell line (Balb/c 3T3 clone A31) (gift from Department of Swine Diseases of the National Veterinary Research Institute in Pulawy) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (ATCC). The media were supplemented with 10% BCS (Balb/c 3T3), 10% FBS (HepG2), 1% L-glutamine, and 1% antibiotic solution. The cells were maintained in 75 cm² cell culture flasks (Nunc, Roskilde, Denmark) in a humidified incubator, NuAire (Plymouth, MN, USA) at 37 °C, in an atmosphere of 5% CO₂. The medium was refreshed every two or three days and the cells were trypsinized by 0.25% trypsin–0.02% EDTA after reaching 70–80% confluence. Single cell suspensions were prepared and adjusted to a density of 2 × 105 cells/mL (HepG2) and 5 × 104 cells/mL (Balb/c 3T3). The cell suspension was transferred to 96-well plates (100 µL/well) and incubated for 24 h before the exposure to the study compounds.

L-aspartic acid (Sigma-Aldrich, UK), 1,4-diaminobutane (DAB) (Sigma-Aldrich, ≥99%), cysteamine (CYSE) (Sigma-Aldrich, UK), cystamine (CYS) (Sigma-Aldrich, UK), dimethylformamide (DMF) (VWR International, USA), dimethylsulfoxide (DMSO) (Sigma-Aldrich), o-phosphoric acid (VWR), imidazole (ACS reagent, ≥99%, Sigma-Aldrich), citric-acid*H₂O (ACS reagent, ≥99.9%, VWR), sodium chloride (99–100.5%, Sigma-Aldrich), phosphate buffer saline (PBS) (Tablet, Sigma), D,L-dithiotreitol (DTT) (Sigma), 5,5 dithio bis-(2-nitrobenzoic acid) (Sigma, ≥98%, USA), L-cystein (Sigma, ≥97%, USA), Humidified incubator (Nuaire, USA), 100 mm tissue culture dishes (Orange Scientific, Belgium), 48 well plates (Sigma-Aldrich, USA), low cell binding 96 well plates (Nunc, Denmark), Eagle’s Medium Alfa minimal essential medium (αMEM) (Gibco, USA), fetal bovine serum (FBS, Gibco, USA), L-glutamine (Gibco, USA), penicillin and streptomycin (Gibco, USA), L-ascorbic acid 2-phosphate (Sigma-Aldrich, USA), beta-glycerophosphate (Sigma-Aldrich, USA), dexamethasone (Sigma-Aldrich, USA), WST-1 [2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] (Roche, Switzerland), Vybrant DiD (Molecular Probes, USA), 2-Amino-2-Methyl-1-Propanol buffer (Sigma-Aldrich, USA), Alkaline Phosphatase Yellow (pNPP) Liquid Substrate System (Sigma-Aldrich, USA).

The human bronchial epithelium cell line (HBE) and NSCLC cell lines A549, H1299, and H1975, were obtained from American Type Culture Collection (ATCC, Manassas, VA, United States). All cell lines were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, United States) containing 10% fetal bovine serum (FBS; Gemini Bio Products, West Sacramento, CA, United States), 100 IU/ml penicillin and 100 μg/ml streptomycin (Solarbio^® Life Sciences, Beijing, China), at 37°C with 5% CO₂ in a humidified incubator (NuAire, Plymouth, MN, United States).