I am culturing MCF-7 cancer cell line in RPMI with phenol red and without phenol red for my experiment. 10% FBS is used. The passage is 42. For the last couple of passages I see that the cells are not properly growing, where I see lot of cell debris and so many very tiny (dotted) like substances. Previously I had never faced this problem. I thought it could be contamination in media and I cultured the media to check it. There were a very few spots. But then I prepared the fresh media and subcultured the cell again. But still having the same problem. i incubated in 5% CO2.
As per you hv told tiny dotted like substance ,recently we hv faced this problem in our lab.We found the cells were not attaching and dying very soon.Otherhand the particles are multiplying indicating contamination.
To solve the problem we hv done contracon of incubator,fumigation of laminarhood and cell culture lab , autoclaved all materials and discarded old media and prepared new media and filtered to use.
If you are in hurry ...you can check your incubator water contamination under microscope and add pinch of sodium azide(Caution:very toxic )in that. or first clean the incubator with methanol-detergent combination then wipe with ethanol and finally do contracon.
then change the water add some CuSo4 in that
@Moyeenul,
It sounds like there is a possible mycoplasma infection. Mycoplasmas, the smallest known free living organisms, are relatively common bacterial contaminants of mammalian cell cultures. Regulatory guidance requires that all products derived from mammalian cell culture be tested for the presence of mycoplasma.
Follow the advice given by Saswat and also go for mycoplasma detection using a commercial PCR based kit (MycoSEQ™ Mycoplasma Detection Kit, cat#4384772, Life Technologies).
In your question you did not mention whether you are regularly adding antibiotics (Pen/Strep or gentamycin)? Using P/S will reduce the chances of possible bacterial contamination into the mammalian cell culture.
I am sharing the protocol for mycoplasma detection as a ready reference.
Good luck!!!
thanks both of you. @ Mandal: yes every time i prepare media using Pen/Strep. But the thing is this problem I am facing only for my cell line. our lab has few more cell lines those are handled by other students but they don't have this problem.
The problem should be the passage, 42 respectively. The cells become senescent. Normally, you stop culturing at passage 35-38. I had the same problem with aged cells (or great number of passages).
In case of contamination you should see a color change of the media (towards light yellow) right the next day of a suspicious manipulation. By classic microscopy you may observe very small round-shaped bodies (20-30 times smaller than a cell) that are "shaking", indicating a bacteria contamination.
Maybe the problem is due to cell senescence for excessive passages. You will save time changing thawing a new cell aliquot from liquid N2
I would also suggest mycoplasma, we had the same issue not too long ago...
Small tiny particles sounds like bacterial contamination with limited antibiotic resistance. Happens from time to time and is the reason why they don't overgrow your culture over night but hang around. Perhaps someone in the lab had a recent infection/sickness? Throw away all cultures and start again with a new frozen stock with fresh media AFTER the hood and incubator have been cleaned properly. If you handled these cells over time the infection will appear in your colleagues cultures as well if you don't clean EVERYTHING (incl for example the microscope surface).
Mycoplasma could also be present but I would disagree that they can be seen using standardized microscopy. Ideally you should routinely use PCR to confirm that your cells are mycoplasma-free once every month. Also if you see debris that sounds not like a mycoplasma problem, which is mostly overlooked BECAUSE normally the cells appear OK. Mycoplasma can be easily prevented by routinely using face masks since 99% of infections come from investigators talking while working at the sterile hood and breathing aerosols (that incl .mycoplasma) into the culture.
MCF7 generally grow like weed and passage numbers over 100 are not exceptional so I think senescence is the least likely explanation.
May be the cell line changes its morphology and the dots inside the cytoplasm may be tiny cytoplasmic vacuoles. You can confirm by TEM.
Hi AKM,
I agree with a previous comment that your passage number is very high (42). For our commercial in-vitro services at iQ Biosciences using cell lines, we do not exceed >20 passages as cells do mutate over time in culture. MCF-7 is a particular cell line that is sensitive to mutation over prolonged culture. Another tip is to not keep re-using the same flask/plate for in-vitro passage purposes.
Best Regards,
Omar
Thank you all for your valuable suggestions. I have overcame the problem by being more careful during culturing to avoid any chance of contamination. even though i faced the same problem afterwards but again I became more careful. It seems to me that avoiding the touch of pipette tips in the flask and other container or anything specially with our finger helped me to overcome. but I will also check your ideas about mycoplasm.
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