Cytotoxicity Assessment of ADSCs on Scaffolds

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)].