Cell culture (fibroblasts L929)
Preparation of the culture medium: Culture medium consisting of 89% DMEM (with high glucose, L-glutamine, phenol red, without sodium pyruvate), 10% FBS, and 1% penicillin and streptomycin solution was prepared.
Cell cultures: Cells were grown in bottles with a capacity of 75 mL, filled with culture medium and placed in an incubator at 37 ° C and 5% CO2. Every 3 days, the medium was changed. To make suspensions with a specific cell density. The cells in the bottles were washed with PBS and then digested with 0.05% trypsin in PBS. After detaching the cells from the wall of the bottle, they were centrifuged. Next, the resulting pellet was resuspended in the medium and diluted to the density required for the test. To determine the appropriate cell density, they were counted on a TC2 Automated Cell Counter Bio-Rad flow cytometer.
Preparation of scaffolds samples for cell cultures: Samples with a diameter of 5 mm and 10 mm were cut from the scaffoldings, respectively, for 96 and 48-well plates. The cut samples were sterilized in 85% ethanol solution, irradiated with UV rays for 30 min on each side.
Non-contact test: Scaffold discs were placed in a 96-well plate. 200 mL of culture medium was added to each well. Samples were mixed, at 37 ° C, for 24 h. Reference samples were wells containing only samples. At the same time, a cell suspension at a density of 1x10^4 cells / well was plated into a second 96-well plate and incubated for 24 h. After this time, the culture medium in the second plate was replaced with samples from the first plate and incubated for 24 h. After this time, the Presto Blue test was performed.
Contact test: Scaffold discs with a diameter of 10 mm were placed in 48-well plates. 0.6 mL of culture medium containing 1x10^4 cells was added to each. The control sample was welled containing only culture medium. Plates were placed in an incubator at 37 ° C, 5% CO2. After 5 h and 1, 2, 3 days, the plates were removed from the incubator and analyzed.