Bme 2

Dissociated prostate cells were resuspended in 80% growth factor‐reduced basement matrix (either Matrigel^®, Corning, 356231; or BME‐2^®, AMSBIO, 3533) and seeded at the concentration of approximately 50,000 cells/ml, by depositing at least six 40 μl drops at the bottom of a non‐tissue culture‐treated plate. Basement matrix domes were left to solidify for 15 min and covered with ENRAD medium—including Egf (50 ng/ml; PeproTech, 315‐09), Noggin (100 ng/ml; PeproTech, 120‐10C), R‐Spondin1 (10% conditioned medium), A83‐01 (200 nM; Tocris, 2393), and dihydrotestosterone (10 nM; Merck, 10300)—supplemented with Y‐27632 (10 μM; Calbiochem, 146986‐50‐7) and ATRA (10 mM; Merck R2625). Organoids were cultured in a standard tissue culture incubator, with medium replacement every 2–3 days. After 6 days from the initial seeding, organoids were imaged with a Leica MZ16F stereomicroscope and organoid forming efficiency was calculated. For subsequent passages, the basement membrane was dissolved using a recovery solution—including Dispase II (1 mg/ml)—and organoids were dissociated to small clumps/single cells as described above, using TrypLE. Following the first passage, organoids were seeded at the concentration of approximately 25,000 cells/ml. Growth factors and small molecules used in this study are described in Appendix Table S1.

Mouse prostate organoids (mPrOs) were generated from prostate glands collected from adult (6–12 months year-old) C57BL/6J wild-type males. Generation and establishment of mPrOs cultures were achieved as previously described [[27] (link), [28] (link), [29] (link)]. Briefly, single cells or small clumps of cells were embedded in growth factor reduced Matrigel® (Corning, 356231) or BME-2® (AMSBIO, 3533) and plated as a 40 μl dome (1000–2000 cells/dome) in a 12-well cell culture plate (3 domes/well). Matrix domes were left to solidify and covered with ENRAD medium including: 50 ng/ml Egf (PeproTech, 315-09), 100 ng/ml Noggin (PeproTech 120-10C), 10% R-Spondin1 (conditioned medium), 200 nM A83-01 (Tocris, 2393) and 10 nM Dihydrotestosterone (DHT, Merck, 10300). Additionally, the medium was supplemented with 10 μM Y-27632 (Calbiochem, 146986-50-7; for 24–48 h after seeding) and with 10 nM ATRA (Merck R2625). Organoids were cultured in a standard tissue culture incubator. Medium was changed every 2–3 days and mPrOs growth was followed by stereoscopic analysis (Leica MZ16F). Organoids were passed once a week by recovering cells using 1 mg/ml Dispase II (ThermoFisher Sci.) and TrypLE (ThermoFisher Sci.), and mechanically dissociating into single cells or small clumps before replating/reseeding.