Methyl thiazolyl tetrazolium (mtt)

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)).

The human umbilical vein endothelial cell line (EAhy926) was cultured in a high glucose Dulbecco’s modified Eagle mediumwith 10% fetal bovine serum (DMEM/10%FBS, Servicebio, G4510, G8001, China). A sample size of 1 × 1 cm² was cut from BIMA grafts and cultured in DMEM/10%FBS at 37°C for 24 h at a density of 2.5 ml/cm². The culture media (leach liquor) were collected and preserved. The media were replaced with leach liquor from the scaffold cultures diluted with DMEM/10%FBS at a ratio of 1:2. The cells were cultured for a further 1, 3, and 5 days at 37°C. A negative control was prepared using DMEM/10%FBS alone. The mitochondrial metabolic (MTT, Servicebio, G4104, China) assay was applied in assessing the cell growth on the scaffold. The optical density at 570 nm was determined using a microplate reader. The cytotoxicity of each protocol was evaluated by calculating the relative growth rate (RGR) to determine the proliferation index (Xu et al., 2017 (link)). RGR = (mean OD for each group)/(mean OD of the negative control) × 100%.

The transfected cardiomyocytes were cultured in 6-well plates for 0, 24, 48, and 72 h and then treated with 20 μl MTT (Servicebio, China) and 5% CO₂ at 37°C for 4 h. The solution was then aspirated, passed through a shaker with 100 μl dimethyl sulfoxide (Sigma, USA), and mixed gently for 10 min to dissolve crystals. The optical density (OD) values were measured with absorbance at 570 nm using a microplate. Cell survival rate was recorded.

HepG2 cells transfected for 1, 2, 3 and 4 days were seeded into 96‐well plates, with 20‐μL 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide,thiazolyl blue tetrazolium bromide (MTT) (Servicebio, Wuhan, China) in each well for further incubation. After 4 hours, the liquid in each well was replaced with 100 μL dimethyl sulphoxide (DMSO). The samples were shaken slightly for 10 minutes. The microplate reader was used to measure absorbance at 490 nm.