I intend to start working on HeLa cells and I read online that, the doubling time of HeLa cells is ~20.67 hrs. But, meanwhile, I was told by a colleague of mine that when HeLa cells grows old (not sure if I am using the correct terminology- 'grow old' or old generation), need about 48 hrs to become double of its number. Why is it so?
I would appreciate all your answers.
Thank you!
Sandip Ghosh
The term I think you are looking for is "reach a high passage number" which means that you have subcultured your cell line many times. You will want to keep track of how many times you have subcultured the cells with which you are working. Also keep track of how many population doublings your cells have gone through. There shouldn't be a large drop-off in HeLa cell population doubling time. They are notoriously vigorous growers and have contaminated many a slower growing culture. It may help to subculture your cells before they become confluent. You will also want to freeze down some seed stocks from early passage cells so you can return back to an earlier passage if something happens to the cells you are working with or if your work is interrupted for a time. Other problems may be mycoplasma contamination of cells, which will slow down cell growth. You might want to try different levels of FBS and see if that makes a difference. Pay attention to the volume of medium that you are putting on the cells, if there is too much you might not be getting very good gas exchange. It would also pay to get a sample of cells from ATCC or other cell bank and start fresh. Here is a short note that might help a bit: https://www.phe-culturecollections.org.uk/media/114565/m219_passage-numbers-explained.pdf
I have found that old Caco-2 cells can vigorously grow by the addition of biotin (vitamin H) at 5 μg/mL. And, I have safely performed the transport study well (please see file; JCB Fucoidan transport).
I am now considering that oldly established cultured-cell line may be mutated in the biotin synthetase gene, or expression of biotin synthetase gene may be repressed by contaminated virus.
Hello Sandip. I have cultured HeLa cells quite a lot (as many of us have). I totally agree with Dr. Hoffman's statements. I would be very surprised if HeLa growth slowed by the amount you describe due to high passage number. In addition to the comments of Drs. Hoffman and Hayakawa, I would add that typically the most labile component of tissue culture medium is the glutamine. Sigma Aldrich had a very nice study reported in their paper catalog in the "old days". If memory serves, glutamine has a halflife of about 30 days at 4C, so if your medium is old, that could slow the doubling time.
Good luck solving your HeLa growth issue.
I agree with Drs. Hoffman's and Whitacre's statements. I would add that different batches of FBS should be pre-screened for their ability to support the growth of Hela cell line. Also, add freshly prepared glutamine solution to the culture medium as the biological effective shelf life of glutamine is 6 weeks.
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