Can anyone guide and help me to promote the growth of human melanoma cell line (SK-MEL-2) and murine melanoma cell line (B16). We bought both of them directly from ATCC 2 months ago. At begining they were grow very well (DMEM media + 10% FBS+ 1% antibiotic and antimycotic) and we prepared about 10 stocks from each. Now we want to thaw and regrow again, but we can not. The cells are not growing at all. TQ
Perhaps try in RPMI. But if you got many passages in DMEM, the cells may have differentiated, as DMEM promotes pigmentation (which however does not automaticallle imply one another!!). Did they change pigmentation or morphology? Then it will be difficult to make them growing as in the beginning, should start from the brand new stock.
We occassionally have this problem with frozen down vials of SK-Mel cell lines (2 and 28). First they don't like to be trypsinised for long, this appears to really affect their viability. We found we have better success getting them out of liquid nitrogen and growing when seeding initially in DMEM + 20% FBS. Hope this helps.
The problem may rely on the way in which stocks were prepared.
Did you check viability of cell after thawing? Other thing, I am not sure about SKMEL2 but B16F10 rapidly produce pigment in DMEM and after couple of days (maybe couple of passages) they undergo terminal differentiation. Thus, even if they are alive they might never regain ability to proliferate. Are youcells in stocks white, grey or black? If black it may never work.
HIgher FBS levels, higher density, growth on 1% gelatin or basement membrane extract are options to consider and have helped us with a variety of melanoma cell lines. Growth in low oxygen levels has also been helpful a few times, for melanocytes for example.
Dear Fellows,
TQVM for all your answers and comments. Yes, my cells changed color to black, and the stock that we prepared were black and not white or grey. Then, what we have to do to prevent bleckening (pigmentation)?
Best regards
Dear Rahman,
You might try to grow them in RPMI as already suggested by Przemyslaw. At least we grow our b16 cell lines in RPMI/10% FCS without any problems. So I guess you can only try if different media and FCS concentration might allow you to rescue your stock. If not you might want to discuss this problem also with ATCC.
b16 when you growth them on F10+FBS remain white and growth without problem. Pigmentation of melanoma (malonocytes) depands on the concetration of tyrosine in medium, in case of suggested media F10
Pigmentation of melanocytes, and of any melanoma cells that are able to differentiate, depends on the tyrosine concentration as Michael Z said, and also on the pH, and on the cell density.
When the cells are highly pigmented they grow more slowly or not at all.
So for fast growth, you can grow them at pH 6.9 to 7, which you can get with RPMI 1640 medium plus 10% CO2, and with 5-10% FCS. Ham's F10 or F12 have very low tyrosine so that's good, but also have low concentrations of basic nutrients (these media are designed for cloning cells from very low density). So you with Ham's you would need to change the medium almost every day for fast-growing cells like B16 sublines. We plate B16 cells at 1x10*4 cells/ml in RPMI/10% CO2 as above, and they will grow 20-30-fold in 3 days or 50-60-fold in 4 days *(change medium on day 3).
You should even be able to rescue them from a low-viability frozen stock this way - just plate more cells to start with (like 10*6 cells per 3-cm dish or well), and after 4-16 hours wash away any that didn't attach (these are dead).
Human melanoma cells can be grown the same way except they generally don't grow as fast as mouse cells
PS, if you can't get 10% CO2, there is a method on our web site to adjust the pH of a given medium - see method 5. And for freezing pigmented cells, method 4.
http://www.sgul.ac.uk/depts/anatomy/pages/dcbm&m.htm
I recently started using B16F1 (3 months ago) but never had a problem freeze-thawing and reviving. I froze them using invitrogen recovery media first by freezing overnight at -80 degrees in Mr. Frosty and then transferring to liquid nitrogen tank. I use DMEM based CGM supplemented with 10 % FBS and 1% Pentstrep.
To evaluate the anti-invasive effect of human malignant melanoma cell lines, SK-MEL-2, Imiquimod treatment decreased in vitro viability of melanoma cells in a concentration-dependent manner. Imiquimod also elicited a concentration-dependent suppression of invasion in both melanoma cell lines and protein was increased in SK-MEL-2.... Jin Young Jung et all (2014) The Effect of Imiquimod on Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Malignant Melanoma Cell Invasion.........
Hi Rahman,
Our group work with both cell lines. In our expierence we culture both in DMEM 10%FBS without antibiotics, they grow very well.
The last year I had a similar problem with Skmel2, when I tried to thaw them, they took too much time to grow. I realized that my stocks which were for a year freezed (-80º) took a hard work to grow. I suggest to control the time you let them freezed and the percentage of Serum in the Freezing Media (I changed to the 90%).
Best regards
Melina
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