Optimizing 22RV1 Cell Growth: Detailed Protocols and Best Practices

22RV1 cells are a human prostate cancer epithelial cell line derived from a xenograft (transplanted into mice that had undergone castration-induced prostate cancer regression followed by regrowth after androgen-dependent CWR22 grafting). This cell line expresses prostate-specific antigen (PSA). Dihydrotestosterone can mildly stimulate cell growth, and Western blot analysis shows that cell lysates react with anti-androgen receptor antibodies. EGF can stimulate cell growth, but TGFβ-1 does not inhibit growth. These cells are tumorigenic in nude mice. Recent studies have shown that 22RV1 cells produce high titers of human retrovirus XMRV (xenotropic murine leukemia virus-related virus).

Characteristics

l Morphology: Epithelial-like cells that grow in a monolayer.

l Doubling time: ~40 hours

l Antigen expression: Prostate-specific antigen (PSA)

l Marker expression: Androgen receptor

l Growth medium: PM150110 with 10% FBS and 1% P/S

Key Points for Culturing 22RV1 Cells

1. Density Dependence: The subculturing ratio should not exceed 1:4.

2. Cell Recovery: Centrifuge to remove DMSO during cell thawing. Plate the cells and allow them to stabilize for two days, ensuring stable incubator conditions. Initially, the cells may adhere loosely in clusters but will eventually spread out into a good monolayer over 4-5 days.

3. Cryopreservation Recovery: The viability of cells post-thawing is low. A cryopreservation medium ratio of 90% serum + 10% DMSO is recommended.

4. Slow Growth: Cells grow slowly and should only reach 90% confluence.

Subculturing Protocol

1. Remove old medium.

2. Add approximately 2 mL PBS, gently agitate to rinse cells, and discard PBS.

3. Add about 1 mL trypsin, gently agitate to ensure all cells are covered.

4. Incubate for 2-3 minutes until cells detach (cells should round up and detach naturally without tapping the flask).

5. Add 3 mL serum-containing medium to stop the trypsinization. Gently pipette up and down to ensure a single-cell suspension.

6. Centrifuge the cell suspension at 1000-1200 rpm (250-300g) for 3-5 minutes. Discard the supernatant.

7. Resuspend cells in fresh medium, mix well, and seed into new flasks as needed. Loosen the cap or use a ventilated cap for culturing.

8. Check incubator CO2, temperature, and water tray.

Media Change Protocol

1. Remove old medium and add new medium along the side of the flask (not directly onto the cells).

2. Change medium every 2-3 days.

Subculturing Ratio and Frequency

When cells reach 80% confluence, subculture at a 1:2 ratio; cells will reach 80% confluence again in 2-3 days.

Subculture at a 1:3 ratio; cells will take more than 3 days to reach 80% confluence.