Nunctm easyflask tm

The cytotoxicity analysis of the electrospun nanofibres membrane was performed using Human Dermal Fibroblasts adult. The cells were grown in DMEM (Dulbecco’s Modified Eagle Medium), supplemented with 10% FBS (Fetal Bovine Serum) and an antibiotic cocktail consisting of 1% (v/v) penicillin-streptomycin and 1% (v/v) non-essential amino acids. The medium was changed every day. The cell culture flasks (NuncTM EasYFlask TM, ThermoFisher Scientific, USA) were incubated at 37 °C in a 95% humidified atmosphere and 5% CO₂ (MCO-5AC CO₂ Incubator, Sanyo). Cells were allowed to grow in four culture flasks to reach 80% confluence and then were trypsinised with 0.25% trypsin solution at 37 °C for 3 min, followed by the addition of fresh medium to neutralize trypsin. The concentration of cells was 1 × 10⁴ cells/cm². After centrifugation (Roto-fix-32A, Hettich, Tuttlingen, Germany) and re-suspension in fresh medium, the viable cells were incubated in the presence of sterilised membrane samples on flat-bottom 96-well plates for 24 and 48 h. After the incubation time, the cell viability was determined using 3-(4,5-Dimethyl-2-thia zolyl)-2,5-diphenyl-2H-tetrazolium bromide (Merck, Darmstadt, Germany) following the MTT assay. Each type of nanofibre membrane was tested in triplicate.

The alginate-matrix based bioink contained homogeneously suspended MG63 cells at a density of 10⁶ cells/ml. First, cryopreserved MG63 (ATCC, CRL^®-1427^TM) cells were quickly thawed at 37 °C and cultured in T-75 flasks (Nunc^TM Easy Flask^TM, Thermo Fisher Scientific) (250,000 cells in 13 ml of media per flask). The media comprised of 90% v/v MEM without L-glutamine (M2279, Sigma Aldrich) and 10% v/v FBS (10438018, Thermo Fisher Scientific) and was changed every 48 hours until 80% confluency. For creating constructs with red-stained cells for fluorescence and brightfield imaging, the culture media in the 80% confluent T-75 flasks was replaced with media containing 0.001% w/v neutral red dye (Sigma Aldrich) followed by incubation of the flasks for 1 h. The dyed or non-dyed cells were passaged using TrypLE select 1X (Thermo Fisher Scientific) and centrifuged at 120 g for 5 min to create a cell pellet. The bioink matrix was 2% w/v sodium alginate (Manugel® GMB, DuPont, Wilmington, DE) solution in PBS (Sigma Aldrich), autoclaved at 16 psi and 121 °C for 30 min (BioClave 16, Benchmark Scientific Inc., Sayreville, NJ) (resultant molecular weight M_n = 67 kDa). The cell pellet was homogeneously suspended in the alginate solution at 1 × 10⁶ cells/ml to formulate the bioink.

Human MCF-7 breast cancer cells were kindly provided by Professor Zbigniew Madeja, Department of Cell Biology, Jagiellonian University, Cracov, Poland. The MCF-7 cell line was cultured in 75 cm² flasks (Nunc^TM EasYFlask^TM, ThermoFisher Scientific, Roskilde, Denmark) in complete Eagle’s Minimum Essential Medium (EMEM) with 10% fetal bovine serum (FBS). The dissociation of MCF-7 adherent cells was performed with Trypsin-EDTA solution and the cells were counted using the TC20 Automated Cell Counter by trypan blue staining. The propolis samples were prepared in EMEM, so that the final concentration of the solvent (70% ethanol) was 0.3%. The final concentrations of all propolis samples were 0.02 µg/mL and 0.04 µg/mL. All reagents and culture media were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).