I am having trouble with C2C12 cells detaching from the well plates after adding conditioned media. Areas on the left and bottom sides of the wells lack cells after switching to the conditioned media. I assume it is due to the media's higher viscosity than PBS as they only dislodged during the media change and not the PBS wash.
Is there any way to prevent the cells from dislodging when I add the media? Any help would be greatly appreciated!
Experiment overview:
1. cells were seeded 24 hours before the media change
2. media aliquoted with single-channel micropipette against the wall.
Hello Reeve Teo ,
I have a couple follow-up questions:
1) Do you typically perform a media change 24-hours after seeding? If so, I would
maybe suggest giving them more time (2 days) to adhere before doing a media change.
2) What is the type/application of the conditioned medium are you using? It may be that the change their expressions and patterns. For example, I was working with a cell line a while ago that was adherent in standard medium with serum. When I switched to serum-free conditioned medium their expression of adhesion molecules changed once the cells began to differentiate.
- It could also be one or more of the conditioned medium components added. Sometimes you can optimize the formula or method for adding components. Some medium components might react with others in the medium. Other times, a certain component might have a more direct affect on the cells themselves rather than the medium. For example, ascorbic acid is not stable long-term in medium and can lead to cell membrane damage. We have to make and add it fresh to the media during every media change to minimize those risks.
This link has further information that might offer some guidance: https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/mammalian-cell-culture/poor-adherent-cell-attachment-troubleshooting
Hope that helps!
Hi Reeve,
I think there's two possible aspects to look at, either physical disturbance from the media change, or the cells are detaching due to something in your conditioned media.
Running the media over down the side of the well is also how I do media changes, and presuming that you do this slowly, it seems to be an unlikely cause of your problem. However, if the cells come loose immediately after a media change, you could try being a little gentler. You could also test this with a media change at 24 hours, but changing to the same media you seeded in rather than changing to your conditioned media.
Alternatively, there could be something in your conditioned media which the cells don't like.
I mostly grow mixed populations of primary cells, and there are always cell types that don't like culturing and will die after a day or two. These dead cells detach. Maybe there's something in the conditioned media that you're adding that the cells don't like.
Try a media change without the conditioned media and see what happens. If it turns out that there's something in the conditioned media that's killing your cells, that'll be tricky trouble shooting, but at least you'll have narrowed it down somewhat!
Best of luck,
Sam
Dear Genesis Marrero ,
Thank you for your help with this issue.
To address your follow-up questions,
1) Yes, I do media changes 24 hours after the initial seeding. I do this because C2C12 cells grow quite rapidly and reach 60% confluency within 2 days. I also want to ensure that they do not differentiate into myotubes as my main focus is to observe how myoblasts react to the conditioned media.
2) I am using human sera-conditioned media. The purpose of my past experiments is to find a concentration of human serum that allows the cell to survive and proliferate.
Strangely, it is not only the conditioned media that experiences this phenomenon. My standard culturing media of 10% Fetal Bovine Serum + 1% Penicillin Streptomycin + DMEM High Glucose. could there be other factors such as my media not being sufficiently warm enough? (37.5 degrees Celsius)
Sincerely,
Reeve Teo
Dear Sam Siljee ,
Thank you for your help as well!
I agree with you that it may either be a physical disturbance or the make-up of my conditioned media. However, I am leaning towards my pipetting being too rough for the cells to handle as the dislodging occurs even in my standard 10% FBS growing media.
This issue has occurred in every single plate experiment I have performed, even with plates from different brands and well numbers.
I am aiming to find the optimal % of human sera in media the C2C12 cells can handle while still proliferating relatively well compared to a similar conc. of FBS media. Fortunately, 2.5% Human and FBS seems to give optimal and comparative results and I have been working with those percentages.
Sincerely,
Reeve Teo
Reeve Teo
Thank you for clarifying! And no, I don’t think your medium not being precisely warmed at 37C when changing the medium would cause that.
If you are observing the cells to be adhered to the wells, under the microscope, before you do the media change but see them detach after a media change, I would agree that the cells probably require gentler pipetting. If you are worried about recovering your cells, you may be able to collect the media with detached cells and then intentionally detach the remaining cells from well plates and centrifuge them to isolate the cell pellets from each well, without losing as many cells. Although, it might be a tedious process when working with well plates as opposed to culture flasks.
You could perhaps coat your plates with poly-d-lysine to increase the chance they stick a bit better. I have also heard collagen works. Best of luck.
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