I am working on H1 human embryonic stem cells and culturing them on matrigel coated plates. On the second day of passage, I could see the media appearing cloudy and plate looks grainy with a lot of speckles under microscope. I would like to know if it is the problem of matrigel, E8 media, bacterial contamination, or some wierd cell differentiation. It would be great if someone who faced similar problem and got resolved gives input. Please find attached microscopy images of healthy and contaminated H1 cells
It is bacterial contamination.
If you found the same problem even when you use different batch of media but the same matrigel, then the problem might be with the matrigel.
Try using a fresh batch of everything including matrigel and see..!
As Holger Fey suggested, throw the current batch of cells.
when you make the fresh batch of media, keep a check plate in all possible combinations for all the reagents you use, like PBS+basal media, basal media alone, growth medium, growth medium+PBS, trypsin (if you used it in the previous experiments) + basal and growth media etc. and incubate them for at least 48 hours to confirm they are not contaminated and then do the actual culture.
If you did not see any contamination in the check plate for any reagent, but still getting contamination in the actual culture, then it is your handling issue..!
Hello
Exactly , this is bacterial contamination.
See links here , It contains many suggestions i hope help you
Good Luck
http://stemcells.nih.gov/staticresources/research/Xeno-free_hESC.pdf
http://www.sigmaaldrich.com/life-science/functional-genomics-and-rnai/shrna/learning-center/hesc-transduction.html
http://www.sciencedirect.com/science/article/pii/S0012160603002562
https://books.google.com.sa/books?id=VA7xDeozZQAC&pg=PA382&lpg=PA382&dq=H1+human+embryonic+stem+cells+and+culturing+them+on+matrigel&source=bl&ots=NXPMJnGyoC&sig=gS-x6z3OT8Ko6yaaZ4Iaz1Z6LIs&hl=ar&sa=X&ved=0CD0Q6AEwBDgKahUKEwjMkOmIjt7GAhUL6xQKHTrXAHw#v=onepage&q=H1%20human%20embryonic%20stem%20cells%20and%20culturing%20them%20on%20matrigel&f=false
http://research.mssm.edu/zwaka/Documents/PMID12426580_Xu-Thomson-2002.pdf
Hello Anil,
If it's the first time that you are getting the contamination then you can follow the above suggested steps. But from your question it appears that you are getting contamination on 2nd passage frequently even after reviving fresh cells again and again. So there might be some contamination issue with culture media or some other culture reagent (FBS also if u r using). I will suggest to go to the root of problem in that case.
Culture some other cells on matrigel so that you can nullify the possiblity of matrigel being a possible contaminant. By culturing the same batch of cells on normal culture plates, you can point out the possiblity tht wheather matrigel is a contaminant or not.
On the other side, grow your cells in normal culture plates (without matrigel) in presence of your currently used growth medium and cocktail, if these cells get contamination , then the problem is with culture media and reagents. I will not advise to through away all culture reagents without proper testing since sometimes thats a costly affair.
n yaa, always throught the contaminated sample as contam as suggested by Mr. Holger as contaminating agent can alter the inherent properties of cell line and hence compromising the authenticity of your results.
Hope it helps !
Most suggest bacterial contamination, but I wouldn't be so sure of it. If this is the second day of culturing and you just remover ROCK inhibitor, you may just have a lot of cell death. To be sure, I would take 1 ul of the media and inoculate a small flask. Unless you see growth in the inoculated media by the next day or the day after, I wouldn't conclude that it is a contamination. The cells, however, do look bad, but I wouldn't make any conclusions until the third or fourth day.
Hi Anil,
Where you able to identify the source of contamination ?
I am having the same issue ( same type of contamination) but can't tell what the organism is, or what the source of contamination, the first time it happened I work on other cell lines that were not affected so I can rule out problems with the aseptic technique. any info will be helpful.
Thank you.
Whenever you find a contamination, you need to go through a systematic process to determine the source. There is no reason to throw away a bottle of media just because you have a contamination in your culture. Take a sample of the media from the bottle and incubate it in an uncoated plate or well. Do the same with a matrigel coated plate. This will at least tell if the media or the matrigel is at fault.
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