Human Organoids

Established patient-derived normal breast organoid cultures [9] were cultured as previously described, within Matrigel (Corning) domes, submerged in medium containing 10% R-Spondin1 conditioned medium, 5 nmol/L Neuregulin 1 (Peprotech, 100–03), 5 ng/mL FGF7 (Peprotech, 100–19), 20 ng/mL FGF10 (Peprotech, 100–26), 5 ng/mL EGF (Peprotech, AF-100–15), 100 ng/mL Noggin (Peprotech, 120–10C), 500 nmol/L A83–01 (Tocris, 2939), 5 μmol/L Y-27632 (Abmole, Y-27632), 500 nmol/L SB202190 (Sigma, S7067), 1 × B27 supplement (Gibco, 17504–44), 1.25 mmol/L N-acetylcysteine (Sigma, A9165), 5 mmol/L nicotinamide (Sigma, N0636), and 50 μg/mL Primocin (Invitrogen, ant-pm-1) in ADF +  +  + . Organoid culture medium was changed every 3 days, and organoids were passed every 5–8 days to avoid confluency. Human MEC derived organoids were treated with pregnancy hormone concentrations same to those utilized for the growth of murine organoids (66.6 ng/mL of 17β-Estradiol, 200 ng/mL of progesterone (Sigma #P8783) and 200 ng/mL of prolactin (Sigma #L4021). We confirmed with qPCR analyses that these grow conditions induced the expression of casein genes, and utilized such analysis to define the collection time points for scRNAseq (untreated cultures, and 10 and 21 after supplementation of medium with pregnancy hormones). Cultured human organoids were processed similarly to mouse organoids prior submission for library preparation and sequencing, with organoids being dissociated with 500 µL of Cell Recovery Solution (Corning® # 354253) for 30 min, followed by incubation with 500 µL of cold Tryp-LE (Thermo Fisher Scientific #12604–013) at 37 °C for 10 min. The dissociated human organoids were likewise resuspended with 1 mL medium, transferred to a 15 mL BSA pre-coated Falcon tube, spun at 300 G for 5 min, resuspended in 1 mL of medium and submitted for library preparation and sequencing.