Hi Naomi,

I think it is because of yeast contamination. If you find the same in a newly thawed vial, then it could be due to improper cell freezing.

I would suggest fumigating the cell culture room and cleaning the incubator with hypochlorite and then wiping it with 70% IPA/70% ethanol.

Add a cocktail of Pen-strep and 50ug/ml Gentamicin, or for fungal conta you can use a cocktail of ketoconazole and miconazole

Hi,

In my opinion, it is not bacterial or yeast contamination, but it is iPSC debris.

Hi Naomi,

I do not think it is contamination, it is most likely cellular debris and the cells seemed to be undergoing stress pretty fast than usual. Try to stain these cells with one of your pluripotent markers. Fiter your complete E8 medium if necessary. additionally try to change the substrate to confirm the presence of theses kind of cells. Addition of 1X antibiotic-antimycotic solution in the complete E8 medium should not affect the morphology of cells.

Hope this helps. All the best!

Naomi Ni Those look like apoptotic cells will cellular contents being expunged out. You can see the transparent circular structures in the second image - these are cell membranes.

Also, do not use Rock Inhibitor more than 24 hours post revival, it might induce differentiation which is why cell morphology looks elongated, and no clear colony boundaries are seen.