I am working with the following melanoma cell lines:
WM115 - MEM supplemented with 10% FBS, 2mM L-glu, 1mM NEAA and 1 mM sodium pyruvate
WM266-4 - MEM supplemented with 10% FBS, 2mM L-glu, 1mM NEAA and 1 mM sodium pyruvate
WM793 - RPMI glutamax supplemented with 10% FBS
1205Lu - RPMI glutamax supplemented with 10% FBS
Sk-mel-28 - MEM supplemented with 10% FBS, 2mM L-glu, 1mM NEAA and 1 mM sodium pyruvate
Sk-mel-5 - Not successful
All lines are growing well in the media indicated above except for the Sk-mel-5 cells which I have not yet successfully revived from liquid nitrogen. The media suggested for these cells (and the Sk-mel-28 cells) in our research Institute culture guidelines (and also the literature) is RPMI supplemented with 10% FBS. I revived both the sk-mel-5 and sk-mel-28 lines on the same day; I did not centrifuge to remove DMSO, I put them straight into pre-warmed RPMI with 10% FBS and incubated overnight. The following morning, I found that neither cell line had attached. I repeated this but centrifuged them to remove DMSO, re-suspended and seeded in RPMI. The following morning, the same results, neither line had attached.
There was a second media indicated for the Sk-mel-28 cells (the MEM listed above) in our in house culture guidelines. I tried reviving both (centrifuging both before seeding) in this MEM. The sk-mel-28 cells came back and have been growing really well the last 3-4 weeks. However, the sk-mel-5 cells did not attach again. In fact, I have not yet successfully revived these cells. I have tried reviving cells frozen down on different dates (2003-2008), in different tanks, by different users, centrifuging and not centrifuging prior to seeding. In light of this, I believe that the media is the problem and that I have not yet found the correct one. I have tried reviving them in all the different medias listed in this post with the exception of the RPMI glutamax. Although trying the RPMI glutamax is next, I thought I would post this in case someone has come up against this or a similar problem with this cell line previously. If you have, any and all feedback would be most welcome.
Hi Alanna,
I even worked with several human melanoma cell lines, and I am agree with you sometimes is frustrating. I would suggest you to test these media. On my hands, they worked perfectly:
- DMEM + 10% v/v FBS, 2 mM L-Gln, 10 mM HEPES, antibiotics 100 U/ml penicillin and 100 μg/ml streptomycin.
- Eagle’s Minimum Essential Medium (EMEM) + 10% v/v FBS, 2 mM Gln, 0.1 mM NEAA, 10 mM HEPES, antibiotics 100 U/ml penicillin and 100 μg/ml streptomycin.
I would also suggest you to take into account to:
- inactivate serum at 56°C for 30 min before culturing cells
- use HEPES to avoid hyper-acidification of the medium, particularly when reviving cells
If these medium do not solve the problem, you could try to thaw cells using 20% v/v FBS. Sometimes, it seems the right way to overcome freezing down.
All the best!
Both cell lines; I found that they were a little bit slow to recover from liquid nitrogen but after 3-4 passages I maintain both lines pretty much as follows; when a T75 is approximately 80-85% confluent, I passage the cells using 3 ml trypsin, quench using 7 ml media and remove the 10 ml using a 10 ml pipette which never collects the entire cell solution. I then pipette in a fresh 10 ml media into the T75 with the residual cell solution. These can reach confluence in around 7 days depending on the residual amount left behind.
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