Gibco™Advanced DMEM/F-12

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	The culture media should not be stored for longer than 2 weeks at 4°C Tissue culture plates should be pre-warmed (overnight at 37°C)

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
The culture media should not be stored for longer than 2 weeks at 4°C Tissue culture plates should be pre-warmed (overnight at 37°C)

Publication protocol

Establishment of human prostate organoid cultures (timing 20 h): Mince human prostate tissue in small pieces (~ 1 – 5 mm3, Fig. 2b) in a 10 cm culture using a scalpel. Digest the tissue overnight in 5 mg/ml Collagenase II with 10 µM Y-27632 in a 15 ml Falcon tube at 37°C on a shaking platform. Use 1 ml of 5 mg/ml Collagenase II per ~ 50 mg minced tissue. Wash once by topping up to 10 ml with adDMEM/F12 +/+/+. Centrifuge at 200 g for 5 min at 4°C. Resuspend pellet in 1 ml TrypLE with 10 µM Y-27632 and digest for approximately 15 min at 37°C. CRITICAL STEP Pipet up and down every 5 min to ensure efficient digestion (P1000 pipet). Wash once by topping up to 10 ml with adDMEM/F12 +/+/+. Centrifuge at 200 g for 5 min at 4°C. Aspirate supernatant and place digested tissue in ice-cold Matrigel and pipette up and down 5 – 10 times to mix. CRITICAL STEP Work quickly to ensure that Matrigel does not solidify before plating. CRITICAL STEP Do not dilute the Matrigel to much to ensure efficient plating. Count cells using hemocytometer and plate approximately 20,000 cells in a 40 µl drop in the middle of one well of a 24-well dish.
CRITICAL STEP Tissue culture plates should be pre-warmed (overnight at 37°C). Place the dish into the 37°C incubator for 15 min to allow the Matrigel to solidify. CRITICAL STEP Place the plate upside down in the incubator to prevent adherence to the plate bottom. Gently pipette 500 µl of pre-warmed (37 °C) human prostate culture medium plus 10 µM Y-27632 into each well. Refresh medium every 2 – 3 days. After 7 days, remove Y-27632 from the medium. CRITICAL STEP Human organoids are split 1:2 every 1 – 2 weeks. This is dependent on density and whether the organoids are luminal- or basal-derived. For instance, the day 7 basal-derived and the day 14 luminal-derived organoids depicted in Fig. 2b are of the size and density to be passaged. Preferred method of splitting for human organoids is with TrypLE. If organoids are small, but the density is high, do not split with TrypLE, but instead use a fire-polished pipette. Human prostate culture medium: Add 1.0 ml B27, 500 µl nicotinamide (1 M in PBS), 125.0 µl N-acetylcysteine (500 mM in PBS), 0.5 µl of EGF (0.5 mg/ml in PBS + 0.1% BSA), 5.0 µl A83-01 (5 mM in DMSO), 50.0 µl Noggin (100 µg/ml in PBS + 0.1% BSA), 50.0 µl R-spondin 1 (500 µg/ml in PBS + 0.1% BSA or 10% conditioned medium), 50.0 µl dihydrotestosterone (1 µM in ethanol), 5.0 µl FGF2 (50 µg/ml in PBS + 0.1% BSA), 5.0 µl FGF10 (0.1 mg/ml in PBS + 0.1% BSA), 5.0 µl prostaglandin E2 (10 mM in DMSO), 16.7 µl SB202190 (30 mM in DMSO) and top up to 50 ml with adDMEM/F12 (containing penicillin/streptomycin, 10 mM Hepes and GlutaMAX 100× diluted). After passaging, Y-27632 is added to the culture medium (e.g. add 5.0 µl of 100 mM to 50 ml human prostate culture medium).


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