2.3. Cell Culture and Cell Seeding

Human G292 osteoblast-like osteosarcoma cell lines (National Cell Bank of Iran, Iran) were expanded in Gibco RPMI 1640 growth medium (Fisher Scientific GmbH, Germany) supplemented with 10% Gibco fetal bovine serum and 1% Gibco penicillin-streptomycin in tissue culture flasks at 37°C with 5% CO₂ using INC246 CO₂ incubator (Memmert GmbH + Co. KG, Germany).

The cells were grown and passaged until about 80% confluency and then detached from tissue culture flasks using trypsin-EDTA, 0.25% trypsin with 1 mM EDTA (Sigma-Aldrich Solutions, Germany). Finally, the cells were seeded onto the scaffolds at passages 4-5, i.e., the number of times the culture has been subcultured, harvested, and reseeded into multiple cell culture flasks. Each scaffold prototype was then placed in wells of 6-well plates. For each sample, a total number of 4 × 10⁶ cells were suspended in 0.5 ml medium and seeded on each silk scaffold within four steps every 20 min. The wells were filled with culture medium to cover the top surface of the scaffolds. In order to prevent occupation of channels by cells, 48 h after cell seeding, the stainless-steel rods were removed. 3D cell culture was carried out for 14 days inside INC246 CO₂ incubator, and media were changed completely every 2 days.