Mc3t3 e1 preosteoblasts

MC3T3-E1 preosteoblasts[30 (link)] (RIKEN Cell Bank, Tsukuba, Japan) were cultured in regular culture media consisting of alpha-modified minimum essential medium (alpha-MEM; Nacalai Tesque) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO, Grand Island, NY, USA) and 1% Antibiotic‐Antimycotic Mixed Stock Solution (Nacalai Tesque) in a humidified atmosphere of 5% CO₂ at 37°C. The cells were trypsinized and seeded onto sample scaffolds at 4 x 10⁴cell /scaffold before each experiment. In all experiments, cells (20 μL of each cell suspension at a suitable cell concentration) were added to each scaffold using a micropipette and incubated in a 5% CO₂ saturated humidity incubator for an hour at 37°C until the cells adhered to the scaffold. Thereafter, each scaffold was transferred to a plate (size suitable for each experiment) and cultured in an appropriate amount of alpha-MEM with medium replacement twice a week.

MC3T3-E1 preosteoblasts (RIKEN cell bank, Tsukuba, Japan) were cultured in alpha-modified minimum essential medium (alpha-MEM; Nacalai Tesque, Kyoto, Japan) with 10% heat-inactivated fetal bovine serum (Biowest, Nuaillé, France) and 1% Antibiotic-Antimycotic Mixed Stock Solution (NacalaiTesque, Kyoto, Japan) at 37 °C with 5% CO₂ and 95% relative humidity. Media were replaced twice a week. Cells up to passage 20 were used.

MC3T3-E1 preosteoblasts [18 (link)] (RIKEN cell bank, Tsukuba, Japan) were cultured in regular culture media consisting of alpha-modified minimum essential medium (alpha-MEM; Nacalai Tesque, Kyoto, Japan), supplemented with 10% heat-inactivated fetal bovine serum (Biowest, Nuaillé, France) and 1% antibiotic-antimycotic mixed stock solution (Nacalai Tesque, Kyoto, Japan) in a humidified atmosphere of 5% CO₂ at 37 °C. Cells up to passage 20 were used. Before the experiments, cells were trypsinized using 0.25% trypsin/EDTA (Sigma, St. Louis, MO, USA), seeded onto scaffolds in 96-well plates at a density of 1 × 10⁴ cells/scaffold, and suspended in 20 µL of media. They were incubated in a CO₂ incubator for an hour at 37 °C and 5% CO₂ saturated humidity until cells adhered to the scaffold. Each scaffold was then transferred to other 96-well plates and cultured by adding 200 µL of alpha-MEM. Media were replaced twice a week.