Pregm medium

The culture and passage of prostate spheres were carried out as described (49 (link)). Dissociated prostate epithelial cells were prepared from mice at 12 weeks of age. To initiate sphere formation, unsorted mouse prostate cells were prepared in PrEGM medium (Lonza) at a density of 5 × 10⁴ cells per ml. Of the cell suspension, 40 ml was mixed with 60 ml cold Matrigel (BD Bioscience), and pipetted around the rim of a well of a 12-well plate and allowed to solidify at 37°C for 30 min. One ml warm PrEGM was then added to each well. The spheres were cultured and monitored for 10 days with 50% medium change every 2 days. To passage spheres, Matrigel was digested by 1 mg/ml dispase solution (StemCell Technologie) for 30 min at 37°C. Digested cultures were collected, pelleted, resuspended, and subjected to sequential digestion by 2 mg/ml type I collagenase (Sigma) for 1 hour and 0.05% Trypsin-EDTA (Invitrogen) for 5 min at 37°C, and then passed through a 27-gauge syringe 5 to 10 times, and filtered through a 40-μm cell strainer. Cells were counted by hemocytometer and replated at the density of 5 × 10⁴ cells per 12-well plate.

The androgen-sensitive LNCaP and LAPC-4 human prostate cancer cell lines were purchased from ATCC (Chicago, IL, USA). Both cell lines were maintained in RPMI 1640 medium, supplemented with 10% (v:v) of fetal bovine serum (FBS), 100 IU/mL of penicillin and 100 μg/mL of streptomycin at 37 °C in a 5% CO₂ incubator. The pre-malignant WPE1-NA22 cell line (ATCC), which mimics a stage of prostatic intra-epithelial neoplasia (PIN), was cultured in keratinocyte serum free medium supplemented with 0.05 mg/ml bovine pituitary extract and 5 ng/ml epidermal growth factor (Invitrogen, Carlsbad, CA). Normal prostate epithelial PrEC cells (Lonza Walkersville, Inc., Walkersville, MD) were cultured in PrEGM medium (Lonza Walkersville, Inc.).

Dissociated cells were incubated in PrEGM medium (Lonza) supplemented with 1:50 B27, 20 ng/mL basic fibroblast growth factor (bFGF) and 40 ng/mL epidermal growth factor (EGF, Corning). Matrigel beds were created in 6 well plates by putting 4 separate drops of Matrigel per well (50 μL Matrigel per drop). Plates were placed in a 37°C CO₂ incubator for 30 min to allow the Matrigel to solidify. For each sample, 100 μL of cell suspension was mixed with 100 μL cold Matrigel, and pipetted on top of the Matrigel bed (50 μL each). The plates were then incubated at 37°C for another 30 min. Warm PrEGM (2.5 mL) was then added to each well. The cells were cultured and monitored for 10–14 days with 50% change of medium every 3 days. For immunostaining, the cells were cultured in 8 well chamber slides. Cells were fixed with 4% paraformaldehyde for 20 minutes and subjected to a standard immunostaining protocol.