Hello Lu Liu ,

From my experience with iPSC, the "contamination" you are refering to looks like cells that are spontaneously differentiating inside your propagating cells.

It's an event that can happen and there's several way to go back to colonies that will stay undifferentiated.

What I would suggest is to remove mechanically by aspiration within the laminar flow hood using a microscope the diffenrentiated cells. What you can do also is to perform a scratching technique prior to media replacement in order to eliminate differentiated cells. Usually, you can perform this the day of the cells passage so that the application of the Gentle Cell Dissociation Reagent (Stem cell) can help in elliminating differentiated cells.

Hope it can helps.

Maxime.

Hi,

Unfortunately, you will never completely remove this problem. The problem you presented is typical for some iPS cell lines.

Take the account of photos which you have shown, only one of the colony looks nice. In this situation, if you don't want to lose a cell line you must do the positive selection. When the percentage of differentiated cells in iPSC culture is between 60-90%, positive selection is highly recommended.

1. You need to mark nice colonies under the microscope.

2. Manually scrape colonies with a nice phenotype.

3. Catch scraped colonies with a nice phenotype and transfer to a new dish.

4. Scaped colonies should be seed in a new dish in very low density.

You have to derive a new iPS cell line from a few colonies with a good phenotype.

Unfortunately, in my opinion, phenotypic instability is a feature of your lineage. So you have to regularly remove "bad" colonies. 1. Scrape the "bad" colonies every day. 2. Use ReLeSR (STEMCELL) at each passage. 3. Use positive selection if the percentage of "bad" colonies is very high.

You should also check the karyotype and pluripotency of your cell line.

Good luck!

Justyna Augustyniak

I would manually pick out the weird looking colonies(using scorning method or. a pippete tip) as much as possible.