Seeding and Culturing Mammary Organoids

Organoids were seeded manually according to our previously published protocols^{3 (link),16 (link),33 (link)}. Briefly, single cells suspended in a 3:4 mixture of Mammocult (StemCell Technologies 05620) and Matrigel (Corning 354234) were deposited around the perimeter of the wells of either 24-well or 96-well plates. The cell suspension was kept on ice throughout the seeding process to prevent the gelation of the Matrigel. To seed organoids in a 96-well plate (Corning 3603), a pipette was used to distribute 5 µL of cell suspension (5 × 10⁵ cells/mL) along the bottom perimeter of each well. Once all mini-rings were generated, plates were incubated at 37 °C and 5% CO₂ for 20 minutes to solidify the Matrigel, and 100 µL of pre-warmed Mammocult medium was added to the center of each well using an epMotion 96 liquid handler (Eppendorf). To generate larger rings (maxi-rings) in 24-well plates (Corning 3527), 70 µL of cell suspension (1.4 × 10⁶ cells/mL) was deposited around the perimeter of each well. Following seeding, the plate was incubated at 37 °C and 5% CO₂ for 45 minutes to solidify the Matrigel, and 1 mL of pre-warmed Mammocult was added to the center of each well. Plates were imaged daily using a Celigo S Imaging Cell Cytometer (Nexcelom) in brightfield mode.