We used two methods for three-dimensional culture of prostate organoids from isolated prostate epithelial cells, corresponding to flotation on top of Matrigel or embedding within Matrigel; the embedding method was used for drug treatment experiments and is described below. For the floating method, prostate epithelial cells were resuspended in prostate organoid culture medium, consisting of: hepatocyte medium supplemented with 10 ng/ml epidermal growth factor (EGF) (Corning #355056), 10 μM Y-27632 (STEMCELL Technologies #07171), 1x glutamax (Gibco #35050), 5% Matrigel (Corning #354234), and 5% charcoal-stripped FBS (Gibco #12676), which had been heat-inactivated at 55°C for 1 hr. After resuspension in prostate organoid medium, 100 – 10,000 dissociated cells were plated into wells of ultra low-attachment 96 well plates (Corning #3474) in the presence of 100 nM DHT for mouse or 10 nM DHT for human (Sigma A-8380). 100 μl of fresh organoid medium was added to the wells every four days, and the medium changed every 12 days for up to one month.
For serial passaging experiments, organoids were passaged at a 1:4 dilution every 1–2 weeks with 0.25% trypsin for 5 minutes at 37°C, followed by mechanical dissociation to nearly single-cell suspensions. Organoids were frozen in complete media with 50% FBS and 10% DMSO. The efficiency of organoid formation was calculated by averaging the number of organoids visible in each well after 7 days of growth using a 10x objective. For statistical analyses, efficiency percentages were arcsin converted to perform unpaired two-tailed Student’s t-tests.
For analyses of androgen withdrawal, organoids were passaged and then cultured for 7–10 days in culture medium in the presence or absence of DHT. For induction of Cre recombinase activity in culture, epithelial cells from un-induced CK8-CreERT2; Ptenflox/flox; KrasLSL-G12D/+; R26R-CAG-YFP mice were sorted based on EpCAM and E-cadherin expression, and cultured until organoid formation was evident. The resulting organoids were passaged, followed by addition of 1 μM 4-OHT on the day after passaging to induce Cre recombination.
A detailed protocol for organoid establishment and culture will be provided on Nature Protocol Exchange immediately following publication.
For serial passaging experiments, organoids were passaged at a 1:4 dilution every 1–2 weeks with 0.25% trypsin for 5 minutes at 37°C, followed by mechanical dissociation to nearly single-cell suspensions. Organoids were frozen in complete media with 50% FBS and 10% DMSO. The efficiency of organoid formation was calculated by averaging the number of organoids visible in each well after 7 days of growth using a 10x objective. For statistical analyses, efficiency percentages were arcsin converted to perform unpaired two-tailed Student’s t-tests.
For analyses of androgen withdrawal, organoids were passaged and then cultured for 7–10 days in culture medium in the presence or absence of DHT. For induction of Cre recombinase activity in culture, epithelial cells from un-induced CK8-CreERT2; Ptenflox/flox; KrasLSL-G12D/+; R26R-CAG-YFP mice were sorted based on EpCAM and E-cadherin expression, and cultured until organoid formation was evident. The resulting organoids were passaged, followed by addition of 1 μM 4-OHT on the day after passaging to induce Cre recombination.
A detailed protocol for organoid establishment and culture will be provided on Nature Protocol Exchange immediately following publication.
