I am working with H1 and H9 human neural stem cells that Iwere differentiated from H1 and H9 hESCs by another lab many years ago to neural stem cells and frozen down and stored in our lab for about 2 years. I have just recently thawed a vial of each line and was working to expand them out so that I would have plenty of stored vials of frozen cells. Upon thawing, they were growing pretty rapidly and seemed to be happy, growing in a nice adherent monolayer. Two weeks ago, I split them 1:6 (lighter than my normal 1:4 split) and I noticed slower proliferation. On Wednesday I had 3 plates of H1 and 4 plates of H9s that were all about 60% confluent and on Thursday when I came in, all the H1s had detached and were floating and going to die. On Friday the H9s started to detach and got down to about 50% confluence. The media pH was not changed, there was no sign of contamination or cloudiness, or particles in the media beyond cell debris. I have also checked the incubator temperature and CO2 levels and all seem fine. It is possible that if there is a power outage that it could take a few minutes for the backup generator to kick on. I am still unsure what could have caused this kind of extreme stress on the cells.
I have also cultured fresh media and spent media and have not observed any color changes or cloudiness in either plate. The spent media contains some cell debris.
At this point, I have discarded these sick cells and have thawed another vial of each (H1 and H9) last Monday. I am not sure if now I am hyperaware of cell death or if the culture I have now is still undergoing a lot of stress, but it seems to me like there are a lot of apoptotic bodies present on the cells and I am concerned about the black dots that can be seen within the cells. Additionally, I have noticed some particles in the culture that have brownian motion but also drift across the view on the scope in a straight-ish line. I have just submitted the spent media for mycoplasma testing and am waiting for the results.
I am unsure if the black dots present within the cells and around them are contamination or if the floating debris is contamination. There is no pH or color change.
Those spots on the cell membranes are sometimes caused by mycoplasma contamination. Bacteria will usually be seen between the cells. Your dots seem to be mostly in the cell membrane. I wouldn't worry to much about one or two odd floaters in the media, this could be any number of things that are not necessarily viable or harmful to your cells. The mycoplasma, on the other hand, will be problematic. You should get a Mycoplasma Detection Kit to test your culture so you can be sure.
If you ruled out bacterial, mycoplasma and yeast contamination, then it is probably plating density. Neural cells and neural stem cells are EXTREMELY sensitive to plating density and will die just because there's not enough of them in one plate.
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In order to see mycoplasma, you need to use fluorescence and 1000x magnification. Those spots are not mycoplasma.
Hello
Yes, those are not mycoplasma, in addition to fluorescence the mycoplasma contamination can be detected by polymerase chain reaction . PCR is easy, sensitive, specific, fast, reliable, efficient and cost effective.
Article Mycoplasma detection by PCR analysis
Have you tested the media for mycoplamsa?
dont rely on microscope observations as it’s subjective. You need a definitive yes or no
You can get mycoplasma testing kits that are faster than pcr. Thermo do a kit you just add media from your culture to the assay and measure on plate reader a simple yes or no result along with controls.
Mycoplasma testing has come back negative. I am beginning to think this is a plating density issue as Yulia Panina mentioned. I'm assuming the black dots are possibly stress granules? Thanks for your help!
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