Cell growth assay in 3D Matrigel culture

Cells were harvested and resuspended in MEM or F12 media containing 1 % FCS. Cells suspension (100 cells in 50 μL) was then mixed with 50 μL of growth factor-reduced Matrigel (Corning, Borre, France) to form a droplet. The droplet was deposited in a well of 6-well plates and incubated for 30 min at 37°C, in 5 % CO₂-humidified atmosphere. The droplet was then incubated in culture medium containing 10 % FCS for 10 days, with culture medium replaced 5 days later. At the end of culture, cells were dissociated from Matrigel with 1 mL dispase 1 U (STEMCELL Technologies) and centrifugated (300 g, 5 min) to remove Matrigel. Cells were resuspended in culture medium and counted with a hemocytometer.