I have been working with the generation of human iPSC-derived kidney organoids following the Takasato protocol for a couple of years, but only recently I started having troubles with the 2D differentiation phase. Specifically, I treat the iPSCs for 4 days with 6 µM or 8 µM CHIR99021, and, starting from late day 5 (after switching to medium with FGF9), only the cells that were treated with 6 µM CHIR start dying (it looks like they start "swelling" and detaching in big colonies). By day 7, cells are all dead. I want to specify that both cells treated with 6 µM and 8 µM CHIR derive from the same iPSC batch (same vial and same culture). Has anyone ever experienced this before? Thank you!
We have experienced unexplained necrosis in our 3D organoids (rather than 2D) in the past. Curiously, it happened after removal of the FGF-9 and reversal to basal medium (they were growing fine up to that point). It ended up being the Hybridoma medium/supplement that you need to add to APEL-2: It was a new batch and we had to re-optimise and change its concentration in the medium, compared to our old batch. Alternatively, you may also want to check your FGF-9 datasheet, as sometimes there are differences from batch to batch in its activity for the same nominal concentration. Most FGF-9 batches activity are within an acceptable range (but you may get the odd one that isn't). Other than that, I would also suggest re-checking the quality of your iPSC stocks (are they healthy post-thaw? / are they still pluripotent or is there a degree of differentiation in your starting material?). IPSCs are notoriously sensitive to culture conditions and need great care to keep plutipotent and healthy.
The bottom line is, you have to change your materials (preferably one by one) to pin this down.
I hope this helps and good luck!
Hi Ioannis, thank you for your answer and your suggestions! Indeed I have started a systematic investigation to identify possible problematic variables. The FGF-9 that I am using comes from the same batch, so I would exclude it. But of course I will know it for sure once I arrive at that step, once I switch from CHIR-containing to FGF9 medium. Thanks again!
Hi Valentina,
I have also experienced almost the same problem as you experienced before. 30% of iPSCs died after treatment of CHIR99021 for 24 hours. Do you have any idea about this problem?
Many thanks and best wishes,
Longsheng
Hi Blerta Stringa and Longsheng Liao
I managed to keep my cells alive after CHIR treatment changing the inital iPSC seeding density (now it's higher) and using half of the medium volume. The thing that is really puzzling for me is that I don't understand why a protocol that has worked for 3 years on the same iPSC line suddenly doesn't work anymore...
Have you looked into mycoplasma? In my experience, if cells start to behave strangely (in particular iPCs), it might be mycoplasma.
Or you are dealing with a subclone that has developed 'resistance'. (Speculations, of course.
Hi Blerta, thanks for the comment! Yes, cells were tested for mycoplasm and resulted negative.
The facility that produces the line has also characterized the batch thoroughly, the cells look quite the same. It is quite frustrating...
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