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WPE-stem cells are loosely adherent and exhibit features characteristic of stem/progenitor cells present in the embryonic urogenital sinus and in adult prostatic epithelium, including p63 and ABCG2.

The cells show high expression of cytokeratin 5 and 14 and MMP-2 but low expression of cytokeratin 18.

They are androgen-independent for growth and survival.

Cells grow in soft agar with a cloning efficiency of 0.9%.

Cells are not tumorigenic in nude mice even at 6 months after injection.

WPE-stem cells were screened for CMV, HBV, HCV, HTLV 1, HTLV 2, HIV 1, HIV 2, JCV, and MoMuLV DNA sequences. Cells were also tested for 25 species of mycoplasma and Acholeplasma laidlawii . Cells tested negative for all of the above. (personal communication from depositor).

Volumes used in this protocol are for 75 cm² flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. Subculture cells before they reach confluence. Do not allow cells to become confluent.

1. Collect spent medium containing viable, floating, single cells, and prostaspheres.
2. Briefly rinse the cell layer with Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS).
3. 3.0 to 4.0 mL of 0.05% Trypsin - 0.53 mM EDTA solution, diluted 1:1 with D-PBS, and place flask in a 37°C incubator for 3 to 5 minutes. Observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 8 minutes).
   Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach.
4. Add 6.0 to 8.0 mL of 0.1% Soybean Trypsin Inhibitor, or 2% fetal bovine serum in D-PBS, as appropriate, add the spent medium containing floating cells, and aspirate cells by gently pipetting.
5. Transfer cell suspension to centrifuge tube and spin at approximately 125 x g for 5 to 7 minutes.
6. Discard supernatant and resuspend cells in fresh serum-free growth medium. Add appropriate aliquots of cell suspension to new pre-coated culture flasks. An inoculum of 2 X 10³ to 4 X 10³ viable cells/cm² is recommended.
7. Place culture vessels in incubators at 37°C. Cells grown under serum-free or reduced serum conditions may not attach strongly during the 24 hours after subculture and should be disturbed as little as possible during that period.

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