I too had the same experience during differentiation of osteoblast.. but not all only I could able to see the square ones.. But I don't have idea regarding this..

Dear RJ Patrick Smith, I think this is an osteogenic lineage  of Adipose tissue Mesenchymal stem cells as these cells during culturing differentiated to the osteogenic lineage and adipogenic lineage

regards

It look like crystals of calcium oxalate, sodium or uric acid, like we can see in urine test or in people's synovial fluid with gout.

This is clearly not an infection. As Matheus stated, these structures are crystals most likely of calcium oxalate that is precipitating out of the medium or for some other cell-mediated reason. I guess that the crystals will be dissolved if they originate from the too concentrated salt in the medium. However, medium change may not dissolve them if they are produced by the cells.

Hi, we did observe these crystals, and could say they were much bigger as we could see then under 10x easily, in the past in our BM-derived mesenchymal stem cell cultures under standard medium conditions. I think it could be a combination of the glutamine supplement we were using, different salt combination already in the medium and also differentiation of the cells. The crystals were sticky on th ecell membranes but a few washes with fresh medium or hepes saline should clear them.

i hope that helps

They look very ordered, so it is likely to be crystals. Check to see if they are birefringent under polarized light. Salt crystals are usually strongly birefringent compared to organic crystals.

Check out this link:https://hamptonresearch.com/documents/growth_101/20.pdf

We had this before and it was crystals from the culture media, not the cells. Have you tried different batches of media/serum?

As everyone else has said, your picture is clearly depicting crystals which for some reason have precipitated out of your culture media. I too had a similar problem a while ago, though I do not know why it happened. In my experiment, however, I only had the square shaped crystals in my media. If it helps, I'm attaching a picture of the crystals I found in my experiment. Upon google searching, I realized that the square/tetrahedron shaped crystals were in fact NaCl crystals. Hope this helps!

Just check that too much media is not evaporating from your cell culture dishes! Sometimes that may also lead to precipitation of salts!

I have also seen similar type of crystal structures in my cell culture of some other type of stem cells. I filtered the medium, and washed the cells well. And this problem was gone.

Hi RJ Patrick Smith,

This is surely crystal! My suggestion, additionaly to those already presented, is to chek the pH of your medium.

I had similar crystals when water evaporated from the tank of CO2 incubator and humidity changed.

As others suggested already: the objects looks like crystal; they might come from the media. Here are my other (brainstorming) thoughts: 1) you may just put as a control an empty flask/Petri dish without cells in your incubator; this may not appear to be 100% conclusive but may help to see if this is related to the cells - or not. 2) Is there enough humidity in the incubator - or is there a chance that the media evaporate too much water and become too concentrated? 3) Did you prewarm the media to dissolve potential crystals, maybe too long storage in a refrigerator supports crystallisation of some salts.

yes, looks like crystals.  chondrogenenic differentiation requires addition of vitamin C. I have seen ascorbate forming cyrstals (but that was when I miscalculated the concentration...)  What kind are you using ? concentration of ascorbic acd ? normally its 100 microM and you should use the ascorbic acid phophate magnesium salt.

Curiously, we have similar crystals when the cells were differentiated in osteogenic cells from adipose derived stem cells. In my opinion, it is some thing wrong with the medium and it seems calcium oxalate! It is not infection!

The above answers are very good. Patrick says the cells are alive and well underneath. Perhaps the cells are tolerating these crystals ? What do you measure in the chondrogenesis assay ? 

As others suggested they could be crystals, a staining with alizarin can tell you if they are calcium compounds (as somebody suggested calcium oxalate), conversely an Alcian blue staining can reveal if they are produced by your ASCs cells after differentiation into chondroblasts.

I did it before, and usually after differentiation you can not see that kind of crystals, rather bigger pieces (clear with a 20x magnification) of cartilaginous matrix, positive to Alcian Blue staining.

These are definitely crystals.  You do not state if you are executing differentiation in multiwell plates or cell culture flasks.  There is evaporation of your medium which is most likely (and faster) in multiwell plates and during long term experiments.  The ecvaporation is highest in the peripheral weels. So you may be able to avoid it by not using these wells in your experments. So monitor the humidity in your incubator diligently.  Crystals may also form in the medium (calcium salts are know for poor solubility) so filtering the medium may also help.  Since others have incountred this also, it would be useful for all, if you find out if the crystals are calcium oxalate. You might sacrifice one well and get a mass spec done on the crystals. Also contact the medium supplier and talk to them about this issue.  If it is to do with the medium they may need to know about it investigate it.  Good luck, and please let everyone know what how you resolve the problem.

We had encountered crystals formation in mollusk cell culture and speculated that they might be analogue of mollusk shell. They looked like your square ones.

It is really interesting that other researches saw square crystals during osteoblast differentiation!

I apologize, but I am not agree with other colleagues. I think that is contamination, why we can not see the cells. I believe that the cells were dead. We only see the cocos.It is condrocyte differeantiation not osteoblast differentiantion, You could do the culture for bacteria and antibiogram as well to made Dapi do see the DNA in this structures!

These are calcium oxalate crystals!! I have these crystals very often in my bacterial cell culture!!

I don't think fungal cells come in such perfect square shapes, and I doubt you got contamination by diatoms. They look like crystals very much. A scale bar in the pictures would be appreciated.

Ideal calcium oxalate crystals

Hi, I also found these crystals in RPE-derived iPSCs culture.

I was checking the media components one by one and, in my case, those crystals are from the Fetal Bovine Serum. I have filtered the FBS before adding to the media and I was able to remove them from the cell culture media.

yes, oxalate crystal deposition I have experienced the same but I have observed this crystal after 21 days of induction medium.

During my research work with MCF-7 cell lines, we have also encountered some of the crystals, but these ones are more pronounced and increase as we increase the concentration of the ruthenium-based complex. I am also interested in knowing what might be going on. We saw this with only one of our complexes, the rest did not form any crystals. Tomo Saric Michael Raghunath RJ Patrick Smith

If possible, maybe should pick one crystal and try to do SC-XRD analysis, it might be the compounds precipitating in high concentrations.

this is Ca oxalate crystal. this crystal is famous in kidney stones patient that has it shape when we examine their urine.