Type I bovine collagen (6 mg/mL; Nutragen, Advanced Biomatrix) was mixed on ice with 10X DMEM (13.8 mg/mL) with or without phenol red, 10X reconstitution buffer (2.25mg/mL [22 mg/mL sodium bicarbonate and 20 mM HEPES in 0.062 N NaOH]), FBS (8.5% v/v) and L-Glutamine (2 mM). After mixing the solution carefully to avoid bubbles, the pH was adjusted to 7.4 with sterile sodium hydroxide. Cells at different concentrations and ratios were added to the solution and gently mixed. Cell concentration was 5 × 105 cells/scaffold for each cell line (1:1 ratio); 5 × 105 and 2.5 × 105 cells/scaffold (2:1 ratio) and 5 × 105 cells/scaffold for one cell line. Four different trials were tested for this group of scaffolds:
Once the solution was homogenous, it was distributed into tissue culture inserts (0.4 μm pore size, 24 mm diameter, VWR) in 6-well plates, using a volume of 2.5 mL/insert. Plates were transferred to a cell culture incubator (37°C, 5% CO2) for 2h to allow polymerization of collagen. Once the constructs were solidified, 3 mL of warmed cell culture medium was added per well (1.5 mL on top of the gel/inside the insert and 1.5 mL outside/under it (i.e., bottom of the well). Plates were maintained in the cell culture incubator and the medium was changed every two days 35 (link)–37 (link).
Once the solution was homogenous, it was distributed into tissue culture inserts (0.4 μm pore size, 24 mm diameter, VWR) in 6-well plates, using a volume of 2.5 mL/insert. Plates were transferred to a cell culture incubator (37°C, 5% CO2) for 2h to allow polymerization of collagen. Once the constructs were solidified, 3 mL of warmed cell culture medium was added per well (1.5 mL on top of the gel/inside the insert and 1.5 mL outside/under it (i.e., bottom of the well). Plates were maintained in the cell culture incubator and the medium was changed every two days 35 (link)–37 (link).
