Hey Guys I split HeLa cells from a confluent T25 flask into 4 T25 flak. The T-25 flask was over confluent and the media was yellow. I changed the media, gave the cells 1XPBS wash and split them in 1:4 ratio. This was done on 14th of November 2022. Now almost 8 days have passed and the cells haven't reached confluency yet. There are few colonies but I could see most cells are yet to adhere properly.
What should I do?
Thanks and Best
Hello V H Krishna Prasad , usually when we dilute HeLa cells we use these dilutions :
1:10 for 4 days (including the day you split them)
1:12 for 5 days
1:2 for one day to the next
That is, if they are just confluent. When they are a bit too confluent, if they were already detaching they might need a bit more time to re-adhere.
Did you try changing your medium 4-5 days after splitting the cells ?
Also, we usually thaw new stocks when our cells are in culture since ~1 month. If your culture is "old" your cells can take more time to grow.
Hope this helps !
Thank you for your reply Bénédicte Giroudot My Culture is around 3 months old. I revived them in the month of September and now December is upon us. Please inform me why old cells would require more time to be revived.
Each time you passage your cells is a stress for them : because you use trypsin to detach them both from the culture flask and from each other. This is especially true for primary cultures, which is not the case here because HeLa are immortalized cells, but anyway this has an effect. Here in my lab, the morphology and homogeneity of HeLa cells after 1 month is a bit different from when we just thawed them.
Also, maybe the trypsin you use is too strong for the cells ? Or if you leave them too long in the trypsin to detach, this is not good for them and can affect their growth speed. If you don't inactivate your trypsin with enough medium, the reason they do not attach after a few days might be because there is some residual trypsin in your flasks. I'm looking for all possibilities here.
Maybe you changed something in the medium ?
Also, when HeLa cells are too confluent they detach from their support : in this case, maybe you could try diluting them more than 1:4 to see whether this changes anything.
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