I'm trying to culturing the human bronchial epithelial cells in DMEM high glucose,10%Fcs,100x p/s...But after thawing them from the first frozen stock in -80C I got granulated and vacuolated dark cells...So do you think the reason behind this a kind of hidden contamination or bad feeding and passing routine(poor culturing that can stress out my cells)!..is it necessary with cell line to know the number of cells to be plated into the flask each time!
If I split my cells till the passage 10 then I frozen few vials THEN I did thawing to one of these vials so what is the number of passage will be 11 or we can restart from one!
Hi Doaa,
It seems like your cells are degraded. This kind of morphology can be observed due to few reasons
1. Too long trypsinization of the cells at cryopreservation
2. Poor cryopreservation techniques
3. Poor thawing practices
Assuming 1 and 2 are properly done (Because they are not under your control now); Let me ask you few questions.
· Did you washed the cell pellet properly to remove the DMSO added in the cryopreservation?
· Did you remove the media at least within 8hrs after culturing the thawed cells in a dish?
DMSO remaining in the media can kill the cells, to avoid this we need to resuspend the pellet twice in media before culture in the plate. Changing the media after few hours will enhance removal of remaining DMSO, cell debris, unattached dying cells and inflammatory cytokines accumulated at thawing process due to the stress.
I would advise you to check the plate 4 hours later under the microscope and see how many cells are floating. If you have more than 80% cells are floating, they are severely stressed at cryopreservation and under necrosis/apoptosis leading to release harmful cytokines to the other cells.
Some experts suggest keeping the discarding media (after 8hrs from the plates containing thawing culture) placing that media in a new plate and incubate for 24-48hrs. This will protect the live cells that take longer time to get adhered to the plate.
Good Luck..!
Hi Doaa,
I completely agree with Susara's answers and that your cell viability isn't that high. Too long trypsinization of your cells can damage them and disrupt their growth, along with too high concentrations of DMSO in your cryopreserve. DMSO can be toxic and inhibit cellular growth.
I've had this problem with my 3T3-L1 cell lines and it's taken a while for them to grow. Typically after freeze thawing, the cells are very sensitive and can die much more easily. I would try being as gentle as possible with the cells.
Normally I thaw the cells in a 37C water bath until half of the ice crystals are melted and then proceed to transfer the media into a T25 flask with pre-warmed (37C) full culture media and gently distribute the cells evenly. After about 4-24 hours your cells should start to adhere to the flask (if adhering cells) and then you can replace the media to remove the DMSO.
Good Luck!
Susara Ruwan Kumara Madduma Hewage
Thanks a lot for your contributions...
Regarding question number one:-
- When I remove cryo tube from - 80C;unscrew cap slightly letting pressure equilibrate then I close cap completely...
- Then in 15 ml conical tube I added 9ml prewarmed warmed media with 1 ml from the thawed cryo medium....I warmed everything at room temperature.
- Then I make centrifugation 5 min...
- After re-suspension of pellet and seeing it in the 25cm flask I changed the medium after 24'1st change'....Then I do second change for the media after reaching 40% Con-fluency...
- I read once before that thawing with centrifugation is usually more stressful for sensitive cells so I tried to do plating for the warmed cry medium directly in the flask but it doesn't work....
Note:I used DMSO 10 % for freezing.
..............................................................
For Andrea Maggioni
You followed
ATCC Animal Cell Lines instructions as I can see...Im using 1x Trypsin-EDTA Really they took long time trypsinization and I feel that im losing them because of this so what is the proper time for this line detach smoothly and freely!!
Thanks for all....
Thanks Andrea I will try not to invent the wheel ANYMORE.
How about the combination of Trypsin-EDTA!...Does it work for these kinds of cells because I don't have right now PVP Polyvenylpyrrolidine (PVP): is sometimes added to trypsin solutions to protect the membranes of sensitive cell lines during trypsinization ...ATTCC
FAQ's
Andrea Maggioni
So you don't perform centrifugation for cells in the thawing process!
which flask to use for culturing of these cells??
and for cryo we should use complete media with pvp??
pvp is suggested from which company?/
i am yet to start culturing the beas 2b cells,, can anybody tell me how to do properly???
which pvp to be used?? there are many given in sigma..which one would u suggest?/
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