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.