Hi Arzoo . Rehman ,

Having higher CO2 due to fluctuations (4-13%) could be detrimental for these cells as higher CO2 will lead to lower/acidic pH and this could be the reason why cells aren't growing normally. Try to stabilize the incubator first and see if that resolves the issue.

Hope this helps.

Hello Arzoo . Rehman

First and foremost, mammalian cells have to be cryopreserved in liquid nitrogen for long-term storage. You may cryopreserve mammalian cells in -80 degree C for short-term storage (not more than 1 year). It has been a long time (almost 2 years) since you have cryopreserved HEK293 in -80 degree C.

Secondly, you are using a CO2 incubator that is fluctuating which is not appropriate for the growth of cells. HEK 293 cells have to be maintained at 5%CO2.

Keeping aside these drawbacks, you should focus on reviving your culture. I would suggest that you centrifuge your cells if they are not adherent, as HEK293 is semi-adherent, at 1000rpm for 5 mins. Discard the supernatant, tap the cell pellet gently and resuspend the cells in 2ml complete growth media. Mix gently and add the entire cell suspension in a well of a 6-well plate. Incubate at 37 degree C at 6% CO2 (5% CO2 would be much better for the survival of cells). If you reduce the growth surface area, the few viable cells (if they still exist) in culture will be able to come in close proximity to each other helping in revival of culture. Hope this will work!

Good luck!

Malcolm Nobre thank you i do understand two issues as you have mentioned but I am working in a compromised situation.

Cells have adhered.

I will keep your advice on growth media next time.

thank you