Rat tail collagen 1 solution

Human mammary fibroblasts were suspended (500,000 cells/mL) in a rat tail collagen I solution (2.2 mg/mL; BD Biosciences, San Jose, CA, USA) as described [64 (link)]. MEM (Invitrogen, Carlsbad, CA, USA) supplemented with 1.8 mg/mL NaHCO₃ (Sigma-Aldrich, St. Louis, MO, USA) and 2.3 mg/mL L-glutamine (Invitrogen) was used as a diluent and pH was adjusted with 0.22 M NaOH. For each replicate, 500 μL cell-collagen mixture was dispensed into a single well of a 24-well plate and incubated at 37°C for 1 hour to facilitate collagen polymerization. Next, the collagen gels were detached from the well using a pipet tip. Culture media (500 μL) was then added and plates were incubated at 37°C in a humidified, 5% CO₂–containing atmosphere. Images of the collagen matrix were taken at the end of contraction time point, and surface area was measured with ImageJ software (NIH).

Multicellular aggregates were generated in hanging drops by creating suspensions of 2000 cells in 25-μl droplets for 48 h to ensure multicellular aggregation, at which point the aggregates were washed with medium, mixed with 2 mg/ml of rat-tail collagen I solution (BD Biosciences), and polymerized at 37 °C in an incubator. Complete medium was added, and the aggregates were imaged under a bright-field microscope 12 h after seeding. Multi-cellular protrusions and single migratory cells were manually counted.