Organoid culture

The surgical tissue of breast cancer were placed in cold AdDF+++ [advanced DMEM/F12 (Sigma, Saint Louis, MO, USA) containing 1 × Glutamax (Invitrogen, Carlsbad, CA, USA), 10 mM HEPES (Invitrogen, Carlsbad, CA, USA) and antibiotics (Sigma, Saint Louis, MO, USA)] and shipped to the laboratory within 20 min on ice. The tissue was cut into 3 mm³ pieces, washed with 5 mL of AdDF+++ and digested in 3 mL medium containing 2 mg/ml collagenase (Sigma, Saint Louis, MO, USA) at 37 °C for 2 h or more. During digestion, pipetting is applied to promote release of the cells in solution. The digested tissue was suspended in 3 ml AdDF+++, then centrifuged at 1300 rpm. If a red precipitate is formed, red blood cells are lysed in 1 mL of red blood cell lysis buffer (Roche, Basel, Switzerland) for 5 min at room temperature and centrifuged at 1300 rpm after adding 3 ml of AdDF +++. Then the pellet was suspended in 12 mg/ml cold Cultrex growth factor reduced BME type 2 (Trevigen, Gaithersburg, MD, USA). 40 μL of BME-cell suspension droplets were added to a preheated 24-well suspension plate (Corning Incorporated, NY, USA) at 37 °C for 30 min. After gelation was completed, 500 μL of medium was added to each well, containing AdDMEM/F12 medium supplemented with B27 supplement [1×, Invitrogen, Carlsbad, CA, USA], Nicotinamide [5 mM, Sigma, Saint Louis, MO, USA], GlutaMax 100x [1x, Invitrogen, Carlsbad, CA, USA], Hepes [10 mM, Invitrogen, Carlsbad, CA, USA], Penicillin/Streptomycin [100U/ml/100 mg/ml, Invitrogen, Carlsbad, CA, USA], R-Spondin 3 [250 ng/ml, R&D Systems, Minneapolis, MN, USA], N-Acetylcysteine [1.25 mM, Sigma, Saint Louis, MO, USA], Noggin [100 ng/ml, Peprotech, Rocky Hill, NJ, USA], Primocin [50 mg/ml, InvivoGen, FGF 10 [20 ng/ml, Peprotech, Rocky Hill, NJ, USA], Neuregulin 1 [5 nM, Peprotech, Rocky Hill, NJ, USA], FGF 7 [5 ng/ml, Peprotech, Rocky Hill, NJ, USA], EGF [5 ng/ml, Peprotech, Rocky Hill, NJ, USA], Y-27632 [5 uM, Abmole, Houston, TX, USA], A83-01 [500 nM, Tocris, Avonmouth, Bristol, UK], SB202190 [500 nM, Sigma, Saint Louis, MO, USA] [13].

The medium was changed every 3 days and passaged every 2 weeks: 1 mL of TrypLE Express (Invitrogen, Carlsbad, CA, USA) was added to the BME-cell suspension droplets, incubation for 20 min at 37 °C, and mechanical blowing. When the number of single cell in the field of view under the microscope reached 90%–95%, the digestion was stopped. 3 mL AdDF+++ was added and centrifugated at 1000 rpm. The pellet was suspended in cold BME and reseeded in the ratio (1:2) as described above.