*2.7. Three-Dimensional Cell Culture in Scaffolds*

Scaffolds were designed by considering their subsequent use in regular 12-well cell culture microplates. First, cells were detached from the original cell culture microplate and counted using the trypan blue dye method. As viable cells possess an intact membrane, trypan blue cannot penetrate them, but as dead cells have an altered membrane the dye can penetrate them. Therefore, trypan blue was added in a cell sample and cell viability was counted using a Neubauber Chamber (Marienfeld-Superior, Lauda-Königshofen, Germany) and an inverted optical microscope. A total of 100,000 cells (MCF-7) or 40,000 (NIH/3T3) in 250 μL cell suspension were placed onto the center of the scaffolds' surface to allow cell attachment. After 3 h of incubation, 1.5 mL of fresh medium was added to cover the scaffold and the cells were incubated for 72 h. Then, the scaffold was placed in a new well to quantify only the cells attached. It was washed with PBS and 1 mL of trypsin was added. After incubation, 2.5 mL of fresh medium was added, and the cell suspension was collected and centrifuged at 1500 rpm for 5 min. Finally, the supernatant was discarded, and the cells were re-suspended and counted.
