Mouse embryonic fibroblasts (SCRC-1049™, ATCC®) were thawed, centrifuged, and suspended in fibroblast proliferating medium (FPM) containing 88% Dulbecco’s Modified Eagle Medium (DMEM, 12100046, Gibco), 10% fetal bovine serum (FBS, 10Bio500, LGC Biotecnologia), 1% non-essential amino acids (MEM NEAA, BR30238-01, LGC Biotecnologia), 1% amino acids (BME, B6766, Sigma), and 1 µL/mL antibiotics (Amikacin sulfate 16.68 µg/µL, Novafarma). Fibroblasts were placed on a culture flask, and cultured in an incubator, under humidified atmosphere of 5% CO2 and at 37°C, until 80% confluence, then were resuspended to be reseeded on placental scaffolds in order to evaluate their potential for recellularization.
Recellularization was performed in 6 days, by dynamic suspension in a 3D RCCS (RCCS-4SC, Synthecon), under 13 r/min. First, 10 placental scaffolds were placed in a 10 mL sterile culture rotating wall vessel (RWV) bioreactor for 48 h, prior to recellularization, to stabilize the biomaterial in the culture environment contending FPM. Afterward, fibroblasts at density of 3 × 106 cells were added to the vessel containing placental scaffolds. Medium addition was performed with a syringe, while emptying bubble in the vessel with another syringe. Closing the sterile valves, vessel was mounted to the rotator base, and placed in 5% CO2, 37°C incubator. Medium was changed every 24 h.
Recellularization was performed in 6 days, by dynamic suspension in a 3D RCCS (RCCS-4SC, Synthecon), under 13 r/min. First, 10 placental scaffolds were placed in a 10 mL sterile culture rotating wall vessel (RWV) bioreactor for 48 h, prior to recellularization, to stabilize the biomaterial in the culture environment contending FPM. Afterward, fibroblasts at density of 3 × 106 cells were added to the vessel containing placental scaffolds. Medium addition was performed with a syringe, while emptying bubble in the vessel with another syringe. Closing the sterile valves, vessel was mounted to the rotator base, and placed in 5% CO2, 37°C incubator. Medium was changed every 24 h.