I'm trying to grow WM-115 but they've all lost their viability after three passages. It'd be great if you can advice me on few things:
1) What incubation condition is favoured for their growth? I cultured them in 37 degrees, 5% CO2. Does anyone think growing them in 34-35 degrees is better?
2) I use 0.01% trypsin-0.5M EDTA solution despite the manufacturers recommendation of 0.25% trypsin-0.03%EDTA. Will the difference is dissociation solution kill the cells?
These are cell lines established in our lab. For ideal culture conditions, please see the following on our website:
http://www.wistar.org/lab/meenhard-herlyn-dvm-dsc/page/melanoma-isolation-media
WM115 can be slow growing so keep them seeded more densely, and increase FBS levels if you see them unhappy (up to 10% FBS). Some people have switched to using DMEM or RPMI instead of our special culture media, this can change cell behavior so if you do so, keep a backup plate with Tu2% while you transition, or as you thaw your cells directly in the other media, monitor attachment closely for a few hours in case you need to make changes to increase survival.
We grow our cells at 37C and 5%CO2, so no changes needed there.
As for trypsinizing, that can impact your cells as well. If you use a more gentle solution, it shouldn't affect the cells; however, if they sit in trypsin solution for too long because they take a while to detach, that's not good either. If the cells don't reattach to the plates within 3h of passing them, you may have a trypsin issue.
Hope this helps.
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