Hi,
I had thawed the 3T3-L1 cells from the liquid nitrogen and culture it in the T75 flask. The cells did only attached about 1 % confluence after a week, and most of the cells were still floating. Now, I wonder what is happening as the others cells in the incubator are growing well. Thus, what I need to do with this cell; wait until reach its confluence, or just discard it? My labmate tell perhaps the cells have died.
Anybody has experienced this? What did you do? Thanks
If adherent cells don't get adherent within 1 to maximum 2 days of defrosting, I don't think they will (I've cultured >30 adherent cell lines and this is the case for all of them).
I don't have experience wit 3T3-L1 cells but we usually defrost our adherent cells in a waterbath at 37°, add medium and transfer to big or small bottle depending on cell number. The next day we change the medium to get rid of the dead (=floating) cells.
If your cells dont attach within a day or two do you really want them in your culture?
I would change the medium on your cells and give them some time. Some of our cells need 2 weeks with medium change every 2-3 days to reach 70% confluency.
Sometimes it also helps to trypsine the cells and put them back in the bottle if they are growing in clumps and react strongly to contact inhibition.
Hi Samihah,
The most important point is to make sure that the cells were frozen using the correct method. Following thawing, if you have cells which are only 1% confluent after a week, they are no longer good to use. You can try thawing another vial and see if the problem persists.
Hi,
Thank you all for the comments. For thawing process, I have applied similar procedures suggested above, except that I centrifuge the 3T3-L1 cells before culture it in the flask. The method applied has succeed for others culture before. I used the cryo cells from my labmates. Thus, I did not sure her technique of cryopreservation. I believe the cells perhaps has something wrong during cryopreservation. Thus, I decided to just discard the cells, and try others. Thanks again.
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