To assess the migratory and invasiveness potential of cells, we measured spatial cell invasion from multicellular cancer spheroids (3D culture) in an ECM. Briefly, cells were seeded in a multiwall 2% Agarose microwells array templated with a Master 3D Petri Dish 96-well arrays at 3000 cells per well (Microtissues Inc., USA) as previously described, 3000 cells per well (25). Following cell seeding, the microwells were incubated with fresh medium to allow spheroid formation for 48 hours. Forty-eight hours after spheroids incubation, the spheroids were harvested and embedded in a solution of 0.25% methyl cellulose in suitable media. Spheroids were then mixed with collagen (rat tail collagen type I, Corning, USA) in neutral pH, seeded in a 24-well plate, and incubated for 30 min at 37°C. After gel stabilization, 300 μl of fresh media was added to provide sufficient media supply. The gel containing the spheroids was gently detached from the well, and images of the spheroids were taken by an inverted fluorescent microscope (Olympus Corporation, Japan, model IX73) every few hours.

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