Hi all, I have problem with my hepG2 cells.
So before covid lockdown, I froze my cells and put it in Mr Frosty in -80°C. I used Cryostor as the freezing media for 1st batch after I received the cells (since I was adviced that HepG2 cells viability is better with Cryostor). Though after that I replaced it with 10%DMSO in FBS due to the lack of Cryostor at that time. The numbers of the the cells I stocked varied between 3 to 3.8 X 10^6 cells per cryovial.
We transferred the tube to LN2 after about 3-4 weeks (we waited other cells to remove it to LN2 together).
And now we want to wake them up, but the HepG2 cells do not attach to the flask the next day after I thawed using standard thawing protocol (from ATCC). They become aggregates and are still floating in the media. Are they dead?
I tried with other tubes from different batches already, but the results are the same.
I wonder whether this is caused by the prolonged time in -80? The other cells are fine when we thaw them.
Or is this because of too many cells per vial? Though I saw other people froze 4 X 10^6 to 1 x 10^7 cells...
Hi,
Did you check the right concentration of DMSO for your cells? maybe your cells are too sensitive for 10%DMSO and it caused dead cells.
Hi Sanaz. Yes, I checked that the DMSO 10% should be fine for HepG2. But in this case, not only the one I froze with DMSO 10%, but also with Cryostor, the cells still do not attach after I thaw
Are you using the same plastic culture dishes that you used before freezing? because some of them are low attachment .
Hi,
We did encounter the same problem in our lab previously.
The reason behind this was either the FBS or the cryo-preservation/thawing process.
Try to check whether the cells are viable or not using trypan blue. If they are dead, I would say that the cells had died during cryo-preservation. However, if they are alive and the numbers are still increasing (but not sticking down) then the reason might be from medium particularly FBS.
Increase the FBS in the media (UP to 20%) could induce the cell to adhere. If FBS is a bit low in ECM proteins, the cell might not attach. Sometimes the quick thaw of FBS might lead to denaturation of lipoproteins and this affects the cell attachment. Try to thaw new vail in fresh media with 20% FBS, if they don't attach within maximum four days, they won't attach.
You know I found that HepG2 are quite strong cancer cells but temperamental, so take care to the frozen procedure and don't use the cells if they are too much confluent and don't freeze more than 2 *10^6 cells/vial. The cell confluency and density during freezing really affect the viability after thawing. Personally I use only 5% of DMSO and 95% complete growth medium
@Sanaz Hassani yes, I use the same type of T25 treated flask and brand with the one I used before. I heard about this problem from other people too, so I also was wondering to try different flask brand or plates. Thank you for your kind insight.
Hi @Batoul Alallam, thanks for your kind info and suggestions. So today is the 4th day after thawing, last time I tried using 20% FBS. Today I finally could see about ~ 50 - 60 % cells attached.
I took out the floating cells and check with trypan blue, some of them are still alive and the other are dead. I guess this problem occured because I froze too many cells in 1 cryovial. Next time I will freeze less than 2 million as you suggest and then check the % of revival.
- Badges
- Science topic