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.