MCF-10A 3D Culture Protocol

Experiments were performed on the MCF-10A cell line purchased from ATCC (Manassas, VA, USA). Cells were cultured in 2D in DMEM/F12 (Life Technologies, Carlsbad, CA, USA) containing 5% horse serum (New Zealand origin, Life Technologies, Carlsbad, CA, USA), 20 ng/mL EGF (Sigma-Aldrich, St. Louis, MO, USA), 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin, 10 μg/mL insulin, 100 U/mL penicillin/streptomycin (all Sigma-Aldrich). Acini from MCF-10A were cultured as previously described [30 (link)]. In brief, single cells were seeded onto a Geltrex (ThermoFisher Scientific, Waltham, MA, USA) bed and cultivated for nine days in EGF-supplemented assay medium (DMEM/F12 Glutamax, 2% horse serum, 5 ng/mL EGF (Sigma-Aldrich, St. Louis, MO, USA), 0.5 μg/mL hydrocortisone, 1 ng/mL cholera toxin, 10 μg/mL insulin, 100 U/mL penicillin/streptomycin), changing the medium every three days. At day nine, EGF was eliminated from the assay medium for further cultivation, changing the medium every three days [30 (link)], while acini cultivation did not exceed 21 days.