Hello guys
I get a cryovial of HEK293 suspension cells which is low viability after thaw, like 37%. I culturing them in a t25 flask now, Will the viability getting high after cell revived? Is there any suggestion for removing those death cells from culture?
Thanks
Hello Liyuan Zhang
After thawing, some cell lines will show low viability if cryopreservation procedure (gradual freezing at- 1°C/min) or thawing process (quick thawing) is not followed properly. So, low viability on the first day in culture will be obvious and this will generate cellular debris. Viability for most cells declines and reaches a nadir at 24 hours post-thaw. Most, if not all, of this decline appears to be due to apoptosis induced by stress . After this time point, cells will slowly begin to recover and enter the exponential growth.
The presence of dead and dying cells in invitro cultures has a negative impact on the growth and activity of neighboring viable cells. So, keep changing the growth media every 24-48 hrs. till you feel that the cells have begun to recover. Use of smaller growth area would be more suitable in such condition. For instance, instead of T-25 flask you could use a 6-well plate which would help in faster recovery of cells.
Hope this will help!
Best.
Generally, live cells should be more dense than dead cells so if you transfer the culture to a falcon tube and allow cells to settle for 5-10 mins, you can remove the supernatant with dead cells.
If you have such a low recovery it suggests a problem in cryopreservation and/or thawing. Is this vial from a company or were they frozen in house? Did you make sure you removed any DMSO by centrifuging the cells prior to plating?
HEK293 grow best at a density of around 5x106 and you should passage/refresh media every 2-3 days.
Hope this helps.
Agree with Malcolm Nobre!
1. change medium every 24hrs, also think about using higher concentration of FBS if still does not work.
2. Transfer cells to smaller culture area and expand gradually.
For HEK293, they are very common, almost every lab has them. You can find them easily, and their viability has essential effects on transduction, so it's better to use new cells.
Marie Fisher thanks for sharing! I centrifuged them at 125xg RT 5mins as ATCC suggested, DMSO should be remove. I think there's problem during freezing or storage, since both 2 vials show low viability after thaw, but another line have over90% viability after thaw.
It sounds like you used the correct protocol for removing the DMSO! What cryopreservation media did you use? A lot of protocols suggest 95% media/5% DMSO but in my experience some cell lines don't fair very well in this and I usually use 95% FBS/5% DMSO.
Also, how you freeze and recover is important-use a Mr Freeze to allow the vials to freeze slowly in the -80oC before transfer to liquid nitrogen. When recovering, I always place the media in the flask in the 37oC incubator for at least 30 min prior to adding the recovered cells.
Also, have you checked there is no contamination/mycoplasma in your cell stocks?
Hope these suggestions help!
Marie Fisher it's Complete growth medium supplemented with 5% (v/v) DMSO. but i am using bambanker in my lab.
I am putting the cryomedia with cells in a T25 flask containing complete media. The next day I give a PBS wash to the flask and change the media and it really works. I used 2.5% DMSO for storage media and it won't affect the cells.
Thanks Manzoor Ahmed , is this the suspension cells you talking? or adherent cells? on the next day you wash it, you mean spin down to remove conditional media then resuspend with pbs?
Liyuan Zhang For adherent cells, you just throw the media and give a PBS wash to the flask and add fresh media. For suspension cells, put the media along with cells into a 15 ml falcon tube, and pellet it down to remove old media. add PBS to it and centrifuge again to remove PBS. Now use a new flask with fresh media and add the cells to it. Hope it will work.
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Virology
- Virus