I have had some cell culture issues over the past couple of weeks that I cannot seem to figure out why my mammalian cell cultures will not grow in our incubator.
We have no successfully thawed new cell lines in another lab so our media, FBS and flasks seem to be working fine. If I bring the cultures back to our incubator, which is stabily at 37 degrees and 5% CO2 ( checked with Fyrite kit multiple times) they die.
Originally the suspended cells would not adhere ( I am working with HEK and HeLa cells which are adherent cell lines). After cleaning out the incubator ( autoclaved parts and wiped the rest with 70% ethanol) some cells began to adhere but then they slowly died off. The cells that adhered did not look happy- they looked blebbed and stressed.
Can you think of a reason I have not thought of? (Again the media, FBS and cell lines all grow fine in another lab, just not in mine).
Any and all suggestions are welcome!
I believe that the cells are dying at the thawing step.
Because the DMSO is very toxic, the cells die very fast if kept in the freezing media for longer than required and if there are less number of cells in the frozen stock you may not recover live cells.
To maximize cell viability at the thawing step, the trick is to dilute the DMSO as fast as possible.
What I do is that as soon I take out the cryovial, I add room temp media directly onto the frozen cells in the cryovial and mix by pipetting.
This rapidly de-freezes the frozen content on the surface. I transfer as much liquid as possible and mix it with 10 ml fresh medium containing FBS. I then repeat the addition of medium, pippet-mixing and transfering the defreezed content to the 10 ml medium till all the content is defreezed. After this I centrifuge to pellet the cells and discard the medium and re-suspend the cell pellet in fresh medium and incubate.
Good luck
Thank you Sudeep for your suggestion. Unfortunately, it is not the thawing step since I can take a culture that is growing fine in another lab and then put it in my incubator and it dies.
Best,
Erin
Hi Erin,
Does the CO2 provided in the incubator works fine? Also, I would suggest you to use a thermometer to check the temperature again. Try and note 3-4 times a day. Match this temperature with the incubator.
Good Luck.
Check your incubator and bio-safety cabinet for contamination, espacially mycoplasma and fungi. As Pratibha suggested temperature and calibrate CO2 level. Most importantly the relative humidity (water) inside incubator and ambient room temperature.
All the best.
Hi Erin,
There are multiple steps at which you can check:
Check if the cell line is viable or no. At times you are not able to revive them if they aren't fine.
Be careful and quick while thawing and make sure the DMSO does not come to RT.
Be gentle while resuspending the cell pellet after centrifugation.
After thawing, take the cell count using trypan blue to confirm if the cells have been thawed properly or not. Dead cells will appear blue.
Check the cell morphology after plating. Live cells will probably appear roundish.
Check your plate after few hours of placing it in the incubator.
Fumigate your culture room and incubator overnight.
Were you ever able to resolve this problem? I have been experiencing the same problem for the last couple of months and have tried a whole bunch of things but the weirdest part is the cells always grow great in someone else's incubator. Any insights would be greatly appreciated!
i m facing the same problem would you solved the problem if yes then plz reply how
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