I can’t say about colon cancer cells, but I had faced a problem like this with primary macrophage cells. I can see particulate material in your cell culture media, and they look same as I evidenced with my culture.

I suspected on quality/age of serum I was using, and I tried ‘clearing’ serum before use by 10K rpm for 10 mins. Since aggregated proteins can activate cells, removal of such heavy particles worked for me with my cells.

Also you can try adding some additional glutamine supplement to your media, it helps.

Best

It's difficult to tell from these images.  Could these be cells migrating away from the colonies?

I am not absolute sure but have seen this in my primary endothelial culture when there is a nutritional deprivation or under stress. It looks like they try to form tubes . But, I have never dealt with cancer cell lines before. Hope some cancer biologists can give us the answer.

As I am working with Lung cancer cell line, for me it seems that there is cell death in your cell culture due to contamination. As the medium haven't change the color so most probably there is some problem regarding contamination. Is it happen for the first time in your cell line? I suggest you to use new medium and be careful about contamination. 

Hi Hamlet

It is hard to tell from the pictures. What you can do is remove this medium, spin it to get ride of cell debris and place only the medium on a new plate inside the incubator - this way if it is a contamination you will see it soon enough.

Since the cell line you are using grows in clusters, these might only be some individual cells that have a different morphology. Maybe if you give them some time and a cluster starts to form you will see that they become similar to the rest of the culture.

Good luck

Hi,

First of all, it does not look like an infection. It seems that these cells are viable and have a fibroblast-like morphology. Possible explanations: 1) The HT-29 cells outside the colonies are migrating (as Dr LoMonaco suggests) and have gained a EMT (Epithelial–mesenchymal transition) state. Why? Have they been subjected to hypoxia, and then reoxygenated (colors of medium is normal). Some of your colonies are big and some are small - are you careful to make sure you have single cells after trypsination?  2) You could have a contamination of another cell line? Is it passage 6 after ATCC purchase or have they been frozen down after x passages before you started to work with them?

I suggest that you take up new cells from the N2(l) and grow them paralell with these ones for comparison. If you get the same problem (or is it a problem?), toss them and purchase new cells to gain full control.

Good luck!

Hi Hamlet,

 I do not know what is going on with your cells, but I know that antibiotics can sometimes cause problems. If they are not necessary you can remove them. Good luck with your cells,

Michele

Again, it is hard to tell from the images.  They could simply be 'lone' cells and their morphology is different because they should be growing in clusters.  Can you increase your seeding density?  It looks like these cells do grow in clusters.  I don't see any giant cells, which would indicate that your colonies as a whole are healthy and not infected.  Do you test for mycoplasma once in a while?

Thank you for your help!!  

I will do the next recommendations that you proposed me:

1. Use new media and filtering the Serum to clear the heavy particles.

2. Add  some additional glutamine supplement to the media.

3. Remove antibiotics from the media. 

4. Test for mycoplasma.

5. If nothing changes, perhaps its only migrating cells.

Regards, 

Hamlet.

Hi hamlet 

Spin serum before filtering it, only filtering may not help; my experience.

Best

Hi,

Based on the images, there is nothing wrong with your culture. They look 'normal'. 

No evidence of contamination.  The indicated structures are sending our what appear to be pseudopods.  Probably a stage in cell division.

Thank you Saurabh Srivastava!!!.

PK Selbo pointed out EMT as a cause.  When I looked at a higher resolution I ahd two impressions: (1) You have a mixed culture or (2) The cells in dense colonies are dead while spread low density colonies are alive.  I agree that you should get a new ATCC stock.

Look like cells are dividing, its not look like a cross contamination, just wait for few more days to see actually what happens.

The indicated structures look like dendritic cells. When cells are collected from tissue there might be a number of minor cell types that persist in culture. If the culture conditions stress these minor participants they will probably disappear.

@ Hamlet:

How is your culture now?

Any improvements?

generally it use to occur with low density seeding.   

Dear Saurabh Srivastava:

Everything is OK. The culture is growing very good, but now I am having  some technical problems to standardize the technique of cytotoxicity with alamar blue. Thank you for your monitoring. 

It is just normal cell culture. No worries!