Hi all,
I was culturing human primary cells with αMEM (prepared from powder according to manufacturer's instruction) on ultra--low attachment binding well plates. The cells were originally from normal culture plates and I dissociated them with Trypsin/EDTA, and put the cell suspension onto the ultra-low binding well plates. According to our hypothesis, these cells are supposed to form spheroids within 1-2 days. The cells clustered together as expected after 2 days, but with the presence of unexpected crystals deposits, both on the bottom of the wells and in the cell clusters.
The medium was prepared with Glutamax as the source of L-Glutamine, and were filtered prior to the addition of FBS. I am pretty sure that I put a big plate of water in the cell culture chamber to make sure the chamber was humidified. What are the possible causes for the crystal formation? And how to get rid of them?
P.S.: The photo below is one of the cell clusters I observed, and you can see clear crystals embedded within the cluster.
Is there any chance that all of the EDTA is not rinsed out. In a different culture context, this paper shows similar crystals. You could try increasing the number of post-trypsin/EDTA rinses. make the first rinse Calcium and madnesium-free.Article "Influence of disodium EDTA on the nucleation and growth of ...
Have you added ascorbate to your medium, it can form crystals if added at too high a concentration. aMEM I think has AA added to it. It does however look like calcium oxalate which would be unexpected however, see image. Test the PH of the medium, it should be around 7 - 7.4 depending on the buffer system used. PH is important for crystal formation. If your water tray is not being routinely filled up, it could be causing the medium to evaporate increasing the concentration of components such as calcium (If it is in the growth medium) increasing the likelyhood for crystal formation.
There is a good Researchgate discussion from 2014 about pH and culture medium crystals. Low (acidic) pH is a problem. pH after adding Glutamax?. https://www.researchgate.net/post/How-do-I-prevent-the-formation-of-crystals-in-cell-culture#view=530cd74ed5a3f2da428b4577
Ellen A G Chernoff Thank you for your paper recommendations, I did not wash the cells after trypsin/EDTA dissociation so that might be the cause, will try to wash the cells with DPBS before seeding again. Regarding the suggestion on pH of the medium, I don't think it was the cause of the issue because the phenol red in the medium wasn't orange-y, so I am pretty confident that it wasn't too acidic. Thanks for info though!! Jack Jordan I don't have ascorbate in the medium, so i think the crystal might really be calcium oxalate... though I suspect there might also be other crystals as well because the big round sphere in my image (at the top of the cell clump as in the image) didn't really resembling calcium oxalate. Will also make sure the water tank is filled, thanks for the suggestion!
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