2.8. Cytotoxicity study using Lactate Dehydrogenase (LDH) assay
The cytotoxic effects and level of toxicity of fabricated 3D scaffolds seeded with DPSCs, considering various coating conditions and inclusion/exclusion of serum in media, were measured by lactate dehydrogenase (LDH) assay for up to two days under proliferation conditions. The LDH kit (CytoTox 96® non-radioactive cytotoxicity assay, Promega) was used and LDH assay was performed according to manufacturer’s instructions in which the number of cytotoxic cells is quantified by measuring cytosolic LDH enzyme leakage into the culture medium as a result of cell membrane damage [37]. Density 3 (4×104 cells/sample) was chosen to compare the DPSCs toxicity of scaffolds. Briefly, 50 µL of cell culture media was collected from 24-well plates after 24 and 48h following DPSC seeding and transferred into a fresh 96-well plate. 50 µL of LDH assay mixture was then added to each well containing the supernatant and incubated at room temperature in dark for 30 minutes. After 30 min, the reaction was stopped with HCl (1 N, 10 vol %) and absorbance values were obtained at 490 nm using a 96-well plate reader (GloMax Discovery microplate reader). DPSCs grown without scaffolds (2D controls) were incubated with lysis solution for 45 min and used as positive controls (100% dead, maximum LDH release control). The cytocompatibility performance of the scaffolds was analysed by using the absorbance of the experimental groups and the negative control group (scaffold only, with no cells). Cytotoxicity data are presented as the average of three replicates. Two different donors were selected to determine the effects of donor type on cytotoxic effects of DPSCs with and without FBS in culture media.