We work for many years with different gynecological cancer cell lines with the usual conditions. Two of these are T47D (breast) and A2780 (ovarium). For a month, this two cell lines are growing very slow.
The growing conditions and the handling are the same as previously. Earlier, I obtained 10-30 million cells from 1 million starting cell number during a week. Now, the cell number is always low, they grow too slow, the controls usually give too week signs. I tried it repeatedly with fresh aliquot (stored in liquid nitrogen, low passage number). Earlier and now, all cells came from the same aliquot seria. There is no infection in the cultures. We use the same medium and experimental tools for many years. Everything is the same as earlier. Any idea?
Hi András,
I don't have an answer for your problem but some suggestions what could still be checked. I experienced a similar problem some time ago.
In case you keep track of that (if you don't you might consider starting it now), check the LOT numbers of the reagents you're using, e.g. the culture medium, any additives you use, washing buffer, .... If you see a relation between change to a new LOT and the start of your problems you could check with the suppliers, if they had notifications for the LOTs concerned.
You said you use the same experimental tools. Does that include pipette tips and other plastics? Same as above: check the LOTs and whether there have been notifications from the supplier.
Last but not least: you could try to find out whether there was an incident with the liquid nitrogen storage.
Good luck finding out what's going on! I know how frustrating it can be...
What if your complete medium has had a decay of an nutrient like your glycine. or another AA... could try to add fresh. .. your cells in cryo freeze could have had a thawl and refreeze this would kill them too.. check you are getting air exghange with your co2 in the incubator caps loosly attached...
I would not spend too much energy looking for what went wrong but to start over fresh to make sure they grow well to study.
Best of luck!
I agree with Charles: Don't spend too much time on looking into what went wrong.
Just make sure that it's not related to your equipment or other supplies. Otherwise you might end up spending a lot of money on a new vial of cells and run into the same problems again.
I think it is not related to the equipments and the chemicals, because....
1. we maintain 10 cell line in more than cell culture 25 flasks in the same time. 9 cell line are kept in the same medium. Due to this amount, there is always fresh medium in the lab, completely fresh things.
2. All the flask are placed on the same termostate with constant conditions
3. still, only 2 cell lines work poorly. There is no problem with the another cell lines (with the same condition, work with the same equipment).
furthermore, I check the suggested conditions for the cells (medium, CO2, etc.), but it seems that we do it well, following the distributor's instruction
It is not necessary, because we throw away these cells and try to grow up from other aliquot.
When we purchase a cell line, after breeding, we usually share the cells to more aliqout and strore in liquid nitrogen. If the passage number becomes high (or a problem appears), we throw away the flask, and we start to growth up a new fresh flask from an other fresh aliqout. The starting samples are infection-free, they dont contain mycoplasma or other microorganisms.
Fair enough, but mycoplasma contamination can occur in liquid nitrogen. So if you just continue chucking away your cultures and take new vials from the same potentially contaminated frozen stock (or even buy a new batch of cells and freeze it in the potentially contaminated tank) you won't solve your problem.
It may happen due to inappropriate preservation of the said cell lines. Mycobacterial infection is also one the cause of such problem. Other causes include degradation of certain constituents present in the growth medium, quality of serum and Co2 supply etc.
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