*5.4. Invasion in Three-Dimensional Spheroid Collagen Gel*

Type I collagen gel (1.2 mg/mL) was prepared as described by [13]. Type I collagen gel solution (5 mL of 2X DMEM, 1 mL 10X HEPES (0.2 M, pH 8.0), and 4 mL type I collagen (3 mg/mL, PureCol, Advanced Biomatrix®)) were prepared and kept on ice before and during experiments. For this assay, 100 μL/well of collagen gel was added in a 48-well plate; it was allowed to polymerize for 30 min at 37 ◦C to form the first layer of the gel. After gel polymerization, the spheroid was embedded into the collagen gel by pipetting it into the second collagen gel layer (100 μL/well). The collagen–spheroid mixtures were then left to polymerize in the cell culture incubator, the 400 μL of serum-free DMEM media were added on top of the wells. After 48 h, the spent media were collected for ELISA assay. Cell migration from spheroids embedded in collagen gels was monitored under an inverted light microscope (Leica DMIL®, Wetzlar, Germany) and photographed at different time points.
