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 mouse prostate organoid cultures (timing 2.5 h): Sacrifice male mouse at minimally 8 weeks of age (maximum tested 2 years). Isolate the urogenital system (Fig. 1a I). Remove seminal vesicles by breaking/cutting blood vessels and connective tissue and making an incision at the base of the urethra (Fig. 1a II, III; for a detailed isolation protocol of the murine prostate see25). Remove the vas deferens by cutting it near the prostate (Fig. 1a IV). Remove the bladder by cutting it near the base of the urethra (Fig. 1a V). Remove remaining vesicles and fat tissue by gentle cutting (Fig. 1a VI). Remove urethra; carefully pull the prostate lobes, so they are no longer attached to the urethra (Fig. 1a VII). CRITICAL STEP The ampullary gland is not considered part of the prostate. The gross anatomy is very similar to prostate. The ampullary gland is located between the two lobes of the anterior prostate (Fig. 1a IX, b). Do not isolate this part. Isolate each lobe individually (anterior prostate (AP), ventral prostate (VP), dorsolateral prostate (DLP)), or continue with the whole prostate (Fig. 1a VII, VIII, 1b). Mince the prostate (lobes) in small pieces (~ 1 mm3) in a 10 cm culture dish using a scalpel. Digest the prostate in 5 mg/ml Collagenase II with 10 µM Y-27632 in a 15 ml Falcon tube for 1 – 1.5 h 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 150 g for 5 min at 4°C. Aspirate supernatant and 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 with a P1000 pipet every 5 min to ensure efficient digestion. Wash once by toping up to 10 ml with adDMEM/F12 +/+/+ and centrifuge at 150 g for 5 min at 4°C. Aspirate supernatant and place digested tissue in ice-cold Matrigel (Matrigel protein concentration ~75%). 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 too much to ensure efficient plating. Count cells using hemocytometer and plate 20,000 cells in a 40 µl drop in the middle of one well of a 24-well dish (Fig. 1c, Table 2). On average one prostate will yield 25 drops. 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) mouse prostate culture medium plus 10 µM Y-27632 into each well. Refresh medium every 2 – 3 days. After 7 days Y-27632 can be removed from the medium.


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