I work on neural differentiation using STEMdiff™ Neural System. And I got stuck at the first stage when using Neural Induction Medium following the EB protocol. Even one week after replated in the 6-well plate(Day 12 of the protocol), I didn't see normal rosettes. Here are the details.
Did anyone have experience with EB protocol? What may cause this to happen? Any help would be appreciated.
this is a very old method of neuralizing hESCs (EBs). Try monlayer differentiation Article Long-Term, Stable Differentiation Of Human Embryonic Stem Ce...
Article Characterization of Three-Dimensional Retinal Tissue Derived...
. With this said, your hESC culture is probably losing pluripotency by the time you harvest colonies for EBs. Try staining duplicate hESC plates (identical passage, cells, colony size) with NANOG and OCT3/4 to see if you have pluripotent hESCs at the time of cell harvesting for aggrewell.
is this the 400 or 800 aggrewells? We are testing aggrewells and found that the suggested plating ratios needed optimisation. With lower cell numbers (1000 cell per microwell?, or 3k cells per well) working better. So perhaps conduct three different cell plating ratio's? Unlike other spheroid or 3D cultures the aggrewells always use the same final volume of 2ml/well. Which means that the seeding density required for each micro-well, (the wells within the well) need a little tweaking - and are not 100% accurate at each cell count, also impacted by actual viability of cells. We found this impacted outcomes. Also the surface area and seeding density for forming spheroids in other cultureware are not comparable (if you just use the same protocol of 2ml, cell count etc) Good Luck!
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Plating