Cell debris on day 4

[Cecilia Reyneri]

Hi all,

First of all thank you so much for creating this space, it is incredibly helpful!

We are starting now to test the protocol from your Nature Protocol paper with Gibco and with one iPS line generated in house by the core facility.

On day 5, you clearly stated in the protocol to abort the differentiation if there is an excess of debris. At day 4 we have definitely more debris compared to the figures in the paper and I was wondering if you think this is too much debris or it is still feasible to continue (I have attached pictures).

If too much, do you have any idea of what might be the reason?

We performed quality controls on the lines before starting (they are undifferentiated but they performed well in the trilineage differentiation, we also checked karyotyping and CNV) and started the differentiation as indicated in the protocol.

Thank you.

Best,

Cecilia

[Abdullah Khan]

Hi Cecilia, 

That's very kind - glad you find it useful. 

- There is quite a lot of debris there, my feeling is that it is likely down to the aggregate size (they're quite large). 

- My suggestion would be to try the AggreWell protocol here: https://groups.google.com/g/morerganoids/c/DsTTcDasDfY (it's the thread in this group about EB/aggregate formation) to control the size a bit better. Your input cell size might vary across iPSCs (it's been consistent in our hands thus far, but I appreciate it might do). 

- ALthough there is a lot of debris, your aggregates look pretty healthy. I think you should embed some to see if they sprout well. The concern with getting this much debris routinely is that you will probably get a lot of variation across experiments, so I'd suggest trying to get the size uniform (which hopefully will control the debris/shredding) using the aggrewell protocol. 

If you're just starting out I'd also suggest looking at the comBO protocol/pre-print. The gel approach there is much simpler so will save you a lot of time/material etc. 

Hope this helps - thanks for tooting! 

Abs