Hi

To understand better, little higher magnification was needed but still, with this, it is sure

1. This is NOT fungal contamination

2. These are looks like a black particle if they are not moving in random motion if they are moving/vibrating then it will be bacterial contamination

3. If we considered them as black particles, mostly, it comes due to bad water in the incubator or laminar flow issue or culturing practice.

4. One of the reasons could be your coating material, matrigel which have used for culturing iPSCs. This problem with matrigel arises mostly when matrigel is not stored properly say temperature issue or very old stock.

I hope it may help you. I expect more answers from other members.

Regards

Saurabh

Dear Alice, according to your statement that it was your first try in culture of ips cells, i guess that either the ips cells have been dissociated by Trypsin instead of Collagenase or they are pipetted harshly. I think that these particles are your ips cells which are entering apoptosis (Anoikis) due to lack of cell-cell interaction needed for their colonal growth. In contrary to other cells, In culture of pluripotent stem cells including ips ones, it must be avoided to make the cells single. You have to use collagenase with the minimum pipettage.

Dear Alice

If you could not distinguish the contamination by organisms morphology you can found it base on medium changes

If medium turned turbid it was bacterial contamination but in this picture, I think the medium is clear, plus the bacteria are really smaller than these particles and have lots of movement in liquid condition

If medium still bright and clear but the color turned yell as soon as you expected these particles should be fungi

But if the medium color and transparency were normal I think these particles are dead cells maybe the Trypsin treatment time was more than usual that cause cells death

maybe your precoating did not correctly do if you did not precoat the plate hiPSCs could not attache properly

Dear colleagues, Thank you for your contributions! From what I could see there was no movement in the medium. The color of the medium turned yellow. In fact I used Trypsin to dissociate the colony. I will try to use collagenase, as @Alireza Naderi Sohi suggested.

Alice Ferreira To me it seems like mycoplasma contaminated culture. Morphologically it doesnt match with any fungal or bacterial type contamination. If you have a mycoplasma detection kit, kindly check once by taking culture supernatant.

It looks like none of your cells are viable. Those are all dead, floating ones. IPSCs will not adhere to the plate if they have been dissociated with Trypsin. Passage your cells with L7 dissociation buffer or cell dissociation buffer and make sure to add Rock inhibitor on 1st day of passage. Also make sure you are gentle when pippetting your cells post spin. Good luck

Hi Alice,

Hope you've resolved this by now, but it looks like you killed all the cells with the trypsin. What looks like a peculiarity in your cell culture is just excess debris along with the dead colonies and single cells.

Personally, I would recommend PBS/EDTA (0.5mM) as a low cost and easy to use dissociation agent. Rock inhibitor would be necessary on thawing but should be avoided in routine maintenance as cells can become dependent on it to maintain their anti-blebbing status.

You can expect a constant finding of debris along with cells in your hiPSC culture, so it is wise to get to know what is what so you don't look past an infection as excess debris.

Good luck!

hi everyone... I have recently encountered the same issue but in a different manner... the day I passaged my ipsc with .5mM EDTA/PBS it was all good n clear. but the next day I could see debris which later increased and covered the whole plate... I really can't understand what should be done... the debris is not moving but increasing... I tried washing gently with DPBS but didn't help at all.

Hi Nelofar,

What kind of coating are you using?

It could be if the PSCs haven't attached properly (or barely). That they wish to continue proliferating but 'dont like' the surface so die, which is why the amount of debris increases, but not exponentially as would be expected by bacterial contamination.

Also as your "colonies" look no more like colonies, this suggests moreso something funky with the coating.

Good luck!

It's a vitronectin coating sir...

this is the fresh image of the plate.... I am not able to understand the reason behind the exponential increase of debris...

Nelofar Karim that looks like contamination to me. I would start with a new vial of cells. Your iPSC colony also looks like an embryoid body is forming. From experience ive never gotten nice colonies to form from them.

What sort of contamination it might be mam, coz particles are not moving but increasing in size and degrading formed IPSC...

Hi Nelofar,

Given that your debris is increasing, although your (poorly) attached cells are not expanding, and the debris doesn't move independently. I would suggest that it is due to improper attachment. Had you given sufficient time to allow coating?

Strangely I have found that sometimes PSCs can grow upwards, which is what seems to be happening. This could be if they are not comfortable to grow outward on the plastic they grow upwards

If it is an infection, I always advice students to identify where has it come from. Can you put 1mL of medium, and whatever solutions you use in culture on a culture plate and place in a separate stove (ideally at 37celcius away from the cell culture) - if there is a bacterial contamination, the next day this will be present in medium without cells.

Should this be the case, as a consequence trash whichever medium/ solution it is. to do so take away from cell culture, ideally add chlorine/ disinfectant tablet then follow your departmental/ institute SOPs for disposal.

Either way as suggested by Heather you will have to start a new vial of cells. Pluripotency is state that needs to be maintained, once compromised there is no going back (unless of course you reprogram said cells into PSCs again)

Good luck,

Cengiz