Excess fusion of embryoid bodies in Phase II media

[Aparajitha Vaidyanathan]

Hi Abs and everyone, 

We have been having some issues lately at the Day 4-5 stage of the organoid protocol. We have been following the ComBO protocol and starting with Aggrewell plates (day -2) to make a set number of aggregates of specific cell number and then collect these for day 0. We then follow the steps for Day 0 where we transfer the EBs to Phase I media in ULA plates and leave untouched for 72 hours. On Day 3 before resuspending in Phase II media, the EBs look larger, separate and spherical. 

On Day 5, before embedding into granular microgel, we see a lot of issues of EB fusion. We have tried distributing the organoids from one well of a 24-well aggrewell 800 plate to 2x 6-well ULA plates to keep them sparse and not packed. We still see a lot of fusion (Photos from Day 5 EBs attached).

We have been using fresh APEL2 media and fresh aliquots of cytokines.

Would really appreciate any advise. We have tried to embed some of these fused EBs and they do not vascularize or engraft any leukemia cells.

Look forward to hearing from you.

Kind regards, 

Aparajitha

[Abdullah Khan]

Hi Aparjitha, 

This is a problem that we haven't encountered ourselves but have heard about from other people. 

The fused EBs are no good you're right. In my experience of troubleshooting this with others, the most likely culprits seem to be: 

(a) Incubator stability - one of the groups I spoke to had this problem solved once they replaced their low O2 incubator speaking to an issue with oxygenation!

(b) 6-well ULA manufacture - 6-well ULAs seem to have a slight depression that makes aggregates/EBs accumulate in the centre, this may be worse with some batches than others. Do you see this? 

(c) IN some instances it might be a sign of poor hiPSC health and growth - might be worth trying with a lower passage/fresh vial for example. 

Sorry I can't be more help in this instance, it's not a problem I have worked through myself only helped others troubleshoot! 

Good luck and thanks for tooting ! 

Abs 

[Aparajitha Vaidyanathan]

Hi Abs, 

Thank you so much for your suggestions and tips to help us troubleshoot this issue.
Based on your suggestions, we believe it could be an oxygenation issue and we are trying to address this atm.

Kind regards, 

Aparajitha

[Abdullah Khan]

Good luck - another thought I had is some lines don't like the single cell dissociation much, so another thing to try is using the original dissociation protocol (using EDTA or RELESR) as opposed to the aggrewell protocol.