Cytotoxicity Evaluation of Leach Liquor

Prior to cell culturing (Liu et al., 2022 (link)), the leaflets were cut into a square shape with 1 cm² × 1 cm² and cultured in high glucose Dulbecco’s Modified Eagle Medium with 10% fetal bovine serum (DMEM/10%FBS, Servicebio, G4510, G8001, China) at 37°C for 24 h at a density of 2.5 ml/cm². Then the leach liquor were collected. Human umbilical vein endothelial cell line (EAhy926) was cultured in DMEM/10%FBS. The medium was replaced by DMEM/10% FBS and leach liquor diluted 1:2. 5,000 cells were seeded in 96 well plates (n = 6) with 200 μl medium. Cells were maintained in culture at 37°C with 5% CO₂ for 1, 3, and 5 days. Negative controls were prepared with DMEM/10% FBS alone. The mitochondrial metabolic (MTT, Servicebio, G4104, China) was used to evaluate cell growth and determine the optical density at 570 nm with a microplate reader (Thermo Scientific, Multiskan Sky). Relative growth rate (RGR) was used to assess cytotoxicity in each group, RGR = (mean OD for each group)/(mean OD of the negative control) × 100% (Xu et al., 2017 (link)).