I am facing a problem in cell culture- Recently our lab shifted to powder based media (DMEM) from liquid media. I am trying to grow an oral cancer cell line (SAS) but I found that my cells are detaching and started floating in the media after one passage. Please suggest me what should I do to solve the issue. So far I have worked on pH of media. I did not found any change after that.
Dear Anuj !
some trouble shooting points may be :
Try and work out on these issues, i hope you can work up with the problem.
Thanks
Dear Anuj,
1.Check the viability of these detached cells. If you find them viable, centrifuge them and culture the cells with less cell numbers.
2. Trying using DMEM/HAM's F-12 nutrient mixture (combination of 2 medium-http://ar.iiarjournals.org/content/34/5/2113.full.pdf)
3. if these doesn't work probably you need to get back to the conditions they were originally cultured in.
4. Use the following procedure to adapt a cell line to a new medium: 1. Subculture the line at a 1:2 split ratio (split the culture in half ) into two vessels. Maintain one with the original medium and continue to subculture these cells for the entire adaptation process. Use a 1:1 mix of the original and new medium in the second vessel. The culture grown in the original medium serves as a reference point as well as a safeguard in case the adapting cells do not survive the process. The low split ratio helps mitigate the stress associated with subculturing as well as with the new medium.
2. Monitor cell growth in the two media and watch for any change in morphology or growth rate. If they are identical, subculture the adapting cells at the next passage with a 1:2 split ratio in a 1:3 medium mix (25% original, 75% new). 3. Monitor the growth rate and morphology of the original and adapting cultures. At the next passage, split the adapting cultures 1:2 in a 1:7 medium mix (12.5% original, 87.5% new). 4. Monitor the growth rate and morphology of the original and adapting cultures. If the cells are identical, then at the next passage split the adapting cells 1:2 in 100% new medium. At this point, the culture should be adapted to the new medium. To confirm complete adaptation to the new medium, perform functional tests on cells derived from the original and new medium. If at any point in the process the adapting culture fails to perform as well as the reference culture, then allow the adapting culture more time and a few more passages in their current medium mix (e.g., 1:3, 1:7, etc.) until they match the reference cells. The same approach can be used to adapt cells to serum-free medium; simply decrease the serum level in the medium by half with each passage until a 0.06% (or lower) serum level is reached. At this point, the cells can be maintained in serum-free medium. If at any point the growth rate declines, then the serum level should be increased to the level where the cells grew normally. In this procedure, start with the “serum-free” medium supplemented with serum so that only the level of serum changes with each passage.
Thank you Purva, Dolly and Thomas. I will try to work on the suggestions.
I think changing the condition of the media is the possible cause, since the cells may adapt to the old liquid medium, because it was happened to me when I changed the FBS company. My advise is to thew new cells from liquid nitrogen (passage zero).
I am also facing the same problem when I have changed the media EMEM (having 10% FBS and Peni-Strep Antibiotic)from with Phenol red to Without Phenol red for MCF-7 cell line.
have you checked for all the compoenents of powdered medium to be same as that of liquid media? Most of the time, powdered media needs to be added with specific growth factors or sometimes glucose too. This was same for DMEM we were using for Caco2 cells. We had powdered media but we had to add glucose while disssolving the powder.
So I guess you might need to check out the composition of media first. I hope this will help you.
I agree with Thomas and Supriya. Especially check the quality of the water used to make the media. Triple distilled water or highly polished equivalent water made by filtration is essential. In either case, the water should never be prepared or stored in metallic containers. Water should be glass distilled not made in a metallic still. very pure distilled water is acidic and very corrosive for metals. It will dissolve metal ions into solution very rapidly. These metal ions will be highly toxic to your cells in culture.
Agree with all the above suggestions. Also check your water quality. You must use culture grade water (Type 1; Resistivity of 18 MΩ cm). Also check water for pyrogens.
Yes I agree with Robert that water should be quartz distilled for all the cell culture purposes.
Hello Anuj,
Powdered media has never been of any problem to cell lines but what you need to see is the nutrient composition of that powdered media like if it contains amino acids or l-glutamine or lacking any important supplement which was present in your previous media but not in the powdered form. If this doesn't work then you might need to change the media and the best alternative can be a-MEM and don't forget to use a good quality FBS (USA Origin).
Secondly, in case of transition from one media to another (it might be solid or liquid forms of same media as they are never same thats why there is so much difference in price) don't put your cells all of a sudden in different media as that can be a great shock to cells. In that case, use new aliquote of cryopreserved cells and grow it in whatever media you want to and don't forget to change the media next day as ignoring the same can result in deadhered and dead cells. If cell stock is not there and running culture is very crucial t you then you can shift on new media in a gradient concentration dependent manner like 80% old +20% new, 50% old+ 50% new, 80% old+ 20% new, 100% new. That will maintain the inherent characteristics of cells.
We are using dd water autoclaved since years and this works very well in any type of cell culture (even in most sophisticated stem cells).
Some cell lines like HepG2, MCF7 are very sensitive to deadherence once they become overconfluent so try to passage them at 70% confluence.
Last but not the list, the most ignored point, please check the expiry of cultureware. I encountered a case where an old culture plate resulted in complete deadherence of cells and we just kept on looking in problems with media,FBS etc. when we used a new plate after identifying the problem it worked very well.
Hope it helps !
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