I thawed some MCF-7 cells 3.5 days ago and seeded them into an untreated T75 flask. The medium used was DMEM with 10% FBS, 1% pen/strep, and 0.1% mycozap, all filtered. According to trypan blue assessment by Countess, I had a total of ~20 million cells in 10mL of media with 89% viability at the time of seeding.
By microscopy, I can see that they aren't adhering; cells are still floating around when I move the flask. Is anything suspect about these? Am I seeding too high a concentration?
Hello Ralph Lim
Yes, the seeding density is too high. You should seed 1 million cells in 10-12 ml medium per T-75 flask.
After 5-6 hrs. cells should begin to attach. You can also leave the cells overnight and check it out. There could be other reasons as well. Sometimes, if the thawing procedure is not performed in a proper manner such situation does arise. During thawing, cells are under stress. So gentle thawing is necessary.
Also, if the stock is old or if freezing of cells is not done in a gradual manner (cooling at -1degree C per min), then too you can face such problem. Also, check for any fluctuation in environmental conditions, like temperature, humidity or pH which may cause cells not to attach.
Best.
Dear Ralph Lim!
For feed seeding density also take a look at this protocol. These cells also require 2mM glutamine and 0.01 mg / ml insulin.
Cell Culture
MCF-7 human breast cancer cells may be seeded in T75 flasks at 1×106 cells/flask in low glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mM glutamine, 0.01 mg/ml insulin and 1% penicillin/streptomycin mix and need to be incubated at 37°C in an atmosphere of 5% CO2. The medium renewal has to be performed 2 times per week, while cells should weekly be passaged at a sub-cultivation ratio of 1:3 (11). An alternative cell culture medium would be DMEM supplemented with antibiotics/antimycotics and 10% FBS (12).
https://ar.iiarjournals.org/content/35/6/3147
Dear Ralf Lim,
I had such a problem with my MSc project. I just used Glutamine as Alexandr explained.
I can see that you still have a few cells which are adhering to the bottom of the flask. My suggestion is to change the media, use PBS to rinse the cells, and use Glutamine. Just be patient and let them grow, this can take time.
Bests
Are you sure you removed DMSO sufficiently after thwaing? Suspending the cells in the culture medium (with FBS) and mild (100g) centrifugation should remove th DMSO (or the like) before plating the gently resuspended cells in fresh medium. Another problem may be your freezing protocol.
Hi Ralph,
Agree with previous recommendations. I just do not understand why you used an untreated flask. In my experince, MCF-7 cells are easy to work with. But, like other adherent cells, they need to be subcultured in tissue culture-treated flasks/dishes or ECM-coated ones.
And looking at the video, they do not look healthy to me. I might be wrong but I see signs of dehydration and shrinking membrane. And also some little black bodies that look suspicious to be contaminations.
You may want to optimize the freezing/thawing ingredient/procedure for future batches too. Let me know to share mine here if you want.
Good luck!
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