My student was trying to grow 3T3 mouse fibroblast cells for transfection experiment. We received a flask from our colleague and the cell grow healthily with the full medium that we prepared (DMEM high glucose + L-glutamine + 10% FBS + Pen/Strep). After a few rounds of subculturing, we stored the cell in (a) full medium + 5% DMSO and (b) 95% FBS + 5% DMSO. Cells were frozen in Mr Frosty overnight before being transfered to liquid nitrogen tank. After a few weeks, we thawed the cell and tried to regrow them in T25. None of the 3T3 cells attached to the flask. Besides, they are growing in suspension. We did trypan blue staining on the cells and they were living cells. After leaving the cells for 2-3 days, the number of cell count increased. The same medium that we prepared earlier on for propagating the cell was used. The same medium when we used on adherent 3T3 cell (given to us in a flask) managed to grow the cell healthily. So, what could have gone wrong?
Many things can go wrong. One is contamination so that the recovered cells are not the same cell line that you thought you froze down. Problems like this are usually not worth troubleshooting to find out exactly what went wrong, it is better to just start fresh with cells that you are SURE are adherent 3T3 fibroblasts, unless the cell line does not matter to you. Many people have had years of research ruined when it was finally discovered that they were using HeLa cells when they thought they were using CHO-K1 or some other line.
Dear Brian, we did restart the culture with a new flask of cells given to us from a different laboratory but the same thing happened when we tried to thaw the cells.
There seems to be a problem with freezing
i hope you are not spinning at high speed for a long time for cellecting cells after trypsinization or for washing after reviving. Usually 1000g for ~3-4 mins is sufficient.
I normally spin at 500g for 5 minutes after trypsinisation or washing.
I would suggest just to start all procedure with fresh cells, and use 10% of DMSO for freezing. Also, can you check whether there were the same flasks before and after freezing?
king hwa, many people thought that freezing down the cells is easy and tend to take it for granted...i have seen post-docs failed to revive their cells after preparing tons of stock...i always advice my student to revive the cells from liquid nitrogen a week after they prepared the stock...yeah, I think you should increase the DMSO to 10%. Full medium or FBS does not make a big different. Are they using the right culture dishes? I really doubt that 3T3 can grow in suspension but I could be wrong.
Check to see whether they are still viable by trypon blue stain and if not, harness the freezing down procedure...
Many of the variables have been listed already by the readers. If you have ruled all of these factors there can only be the difference in the culture flask (brand, material etc.) that you normally use and that was received with the original culture. Please do check this. It may sound a trivial factor, but can be crucial if the different brands are having different cell adhesion factors that may or may not favorably impact on cryo-storage of the cell lines. Other than this increasing DMSO to 10% is definitely a major option to try out. Hope this helps.
Dear everyone, thank you so much for your comments and suggestions. I will get back to all of you again after i have figured out the exact problem. Cheers!
DMSO is critical of course. Here is an alternative: CO2 is also critical for plating, please check CO2 level, water in incubator, and ask the other laboratory whether they were grown at 5 or 10% CO2.
Dear Artur, we started 2 batches of new flasks of 3T3 cells given from a different lab working on 3T3. They have the 3T3 morphology before we stored them in Liquid nitrogen tank. Now we suspect that LN may have leaked into the cryovial as these vials may have been submerged in the LN instead of preserved using the vapor phase!!! Will get back to everyone after we have sorted the issue. Many thanks for your comment!
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