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WPE1-NB14 cells belong to a family of cell lines, referred to as the MNU cell lines, which are all derived from RWPE-1 cells after exposure to MNU. The larger family of cell lines, including RWPE-1 cells with a common lineage, mimics multiple steps in progression from normal epithelium to prostatic intra-epithelial neoplasia, and then to invasive cancer. The MNU cell lines, in order of increasing malignancy are: WPE1-NA22 (ATCC CRL-2849), WPE1-NB14 (ATCC CRL-2850), WPE1-NB11 (ATCC CRL-2851), and WPE1-NB26 (ATCC CRL-2852).

WPE1-NB14 cells show moderate invasive ability in the in vitro Boyden chamber invasion assay RefWebber MM, et al. Human cell lines as an in vitro/in vivo model for prostate carcinogenesis and progression. Prostate 47: 1-13, 2001. PubMed: 11304724.

The colony forming efficiency (CFE) of 1.85% and the invasive ability of WPE1-NB14 cells are both greater than those of WPE1-NA22 cells (ATCC CRL-2849) RefWebber MM, et al. Human cell lines as an in vitro/in vivo model for prostate carcinogenesis and progression. Prostate 47: 1-13, 2001. PubMed: 11304724.

The cells form small tumors after subcutaneous injection. The tumors are a little larger than those formed by WPE1-NA22 cells (ATCC CRL-2849).

The depositor report that the parent RWPE-1 cell line (ATCC CRL-11609) was screened for Hepatitis B and C, and human immunodeficiency viruses, and was found to be negative.

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

1. Remove and discard culture medium.
2. Briefly rinse the cell layer with Ca++/Mg++ free Dulbecco's phosphate-buffered saline (D-PBS).
3. Add 2.0 to 3.0 mL (to a T-25 flask) or 3.0 to 4.0 ml (to a T-75 flask) of 0.05% Trypsin - 0.53 mM EDTA solution, diluted 1:1 with D-PBS, and place flask in a 37°C incubator for 5 to 8 minutes. Observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 10 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, 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 culture vessels. An inoculum of 2 x 10⁴ to 4 x 10⁴ viable cells/cm² is recommended.
7. Incubate cultures at 37°C. We recommend that you maintain cultures at a cell concentration between 4 x 10³ and 8 x 10⁴ cells/cm².

Note: 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.

Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a Manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994.

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