Perform all steps under aseptic conditions.
- Coating culture vessels with matrigel (optional)
1.1 Prepare 300 µg/ml matrigel solution in phenol red-free DMEM.
1.2 Add 2 mL of solution to T-75 flask.
1.3 Incubate for 1 hour at room temperature.
1.4 Remove excess matrigel.
1.5 Warm to 37ᵒC for immediately use or store at 4ᵒC.
- Thawing frozen VCaP cells
2.1 Warm culture media and T-25 flask to 37°C.
2.2 Rapidly thaw vial in 37°C water bath.
2.3 Transfer cell suspension to a 15 mL conical tube containing 9 mL culture media.
2.4 Centrifuge at 300 g for 5 minutes.
2.5 Resuspend the cell pellet in 5 mL media and seed onto a T-25 culture flask.
2.6 Place flask in humidified incubator set to 37°C and 5% CO2
- Subculturing VCaP cells
3.1 Warm culture media and T-75 flask to 37°C.
3.2 Remove old culture media. Take 1 mL old media and set aside to add to new flask, use the rest to resuspend cells after exposure to trypsin.
3.3 Rinse flask with PBS.
3.4 Add 2 mL Trypsin/EDTA and place flask back in 37°C incubator until detached from flask.
3.5 Suspend cell/trypsin mixture in the old culture media and transfer to 15 mL conical tube.
3.6 Centrifuge cell suspension (300 g for 5 minutes) and collect cell pellet.
3.7 Resuspend the cell pellet in fresh culture media.
3.8 Split cells at a ratio of 1:3 or 1:2 depending on the initial density.
3.9 Add 1 mL old culture media to the new flask.
3.10 Incubate at 37°C.
a. VCaP cells are delicate. Take care to always treat them gently, i.e., slow centrifuge speeds, don’t pipette aggressively, etc.
b. VCaP cells can grow as a monolayer and in floating or adherent clusters (Figures 1 & 2). Cell clumps will likely be present and it may be difficult to break up all of them. Our lab has found that it is better to leave them in small clumps than to over-pipette them.
c. Floating cell clusters and heavy debris are normal characteristics of VCaP cells. Do not discard the floating cells clumps; gently centrifuge cells suspended in media and add them back to parental flask.
d. Our lab has found that coating culture flasks with matrigel can improve adherence and reduce the amount of floating cells (see protocol step 1).
e. VCaP cells grow very slowly. It may take 2-3 weeks for a T-75 flask to reach confluency.
f. VCaP cells should always be cultured with conditioned media (for T-75 flasks: 9mL fresh media + 1mL old media). Conditioned media can be saved and stored at -20°C for future use.
g. VCaP cells are very sensitive to trypsin. Do not subculture them in media containing trypsin.
h. Consult "troubleshooting table":http://www.nature.com/protocolexchange/system/uploads/3831/original/VCaP_troubleshooting.jpg?1439159651 for more VCaP culture suggestions.