I am producing embroïd bodies derived from hiPSC to generate brain organoids by the self-aggregating method following the protocole published by Lancaster in 2013 and 2017. Briefly, I seed 9000 hiPSC (passage 18 or more) dissociated into single cells into low-adherence 96-well plates (that I made myself by coating with poly-HEMA) in hESC media supplementaded with RI and low concentration of bFGF. With some hiPSC lines, I obtained spheroid embryoid bodies. But with some other hiPSC lines, I often obtained ellipsoid embryoid bodies that are bigger and lead to poor efficiency for brain organoid production. All hiPSC lines were validated for multipotency, were tested negative for mycoplasma and were karyotipically normal.
Does other people encountered similar problems when producing embryoid bodies? How can I make embryoid bodies that are more spherical and homogeneous using low-adherent 96 well plates?
Hey Thiery,
This is unusual. I will normally get a few that are not spherical in a full 96 well plate, however most of them are fine and look like good EBs.
The only difference is I use commercially available low attachment plates, which may make a difference. I would recommend asking a company near you for a sample of their 96-well low attachment plates and give them a go. see if you still get this issue?
The only other thing i can think of is debris. Sometimes you can get cotton from stripettes and other sterile contaminants in to your wells, and the iPSC love clinging to them and making more elongated EBs. But i would be surprised if this was happening consistently.
Finally EBs and Organoids are a little more variable in general than 2D cultures, and so you will probably never get a full plate of EBs ALL looking amazing and all differentiating exactly how you want. Definitely make more than you need and then use QC to make sure they are what you were looking to culture. :)
Hope this helps.
Best,
Christopher Lovejoy
Hi Christopher,
Thanks a lot for your answer. I will try with commercial low-attachment plates!
Indeed, in normal embryoid bodie (EB) batchs, some EB are not spherical, but it is only a few of them, like you said. With my problematic hiPSCs lines, the EBs are systematically ellipsoïd and bigger, and even if they formed an ectoderm, they degenerate unpredictably after a few days. These problematic hiPSC lines were however able to form spherical EB when cultivated in Aggrewell in Neural induction medium following the protocol from STEMCELL to generate 2-D neuronal precursors. So the problem seem to affect only specific hiPSC lines, even if these hiPSC sseemed to be of good quality for differentiation experiments...
I also usually observed the debris that you mentionned, but only in one or two well of 96 wells, like you said.
Another option is that I use Aggrewell (from STEMCELL), but I think it would be difficult to select embryoid bodies for quality control and make good brain organoids...
Hi Nicolas,
Thanks for your answer! I am using 96 wells U-bottom plates that I coat with poly-HEMA to make them non-adherent. It work well for some hiPSCs lines, but not for others. I will try with commercial low-attachment plates.
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