Hi all,
I am currently working on differentiating iPSC to neurons. A critical step of the process is dissociation of Neuronal Spheroids.
I am using 0.05% Trypsin-EDTA, 37℃ for 20 mins, then stop with culture medium, centrifuge and dilute in 1ml culture medium+ ROCK inhibitor, mechanically pipetting slowly for around 7 minutes. Still I saw ineffeicient dissociation, some cells still in clumps. Also as some clumps remain undissociated, the cells yield is low. The viability was quite high though(>95%).
So my question is,
- Are you using some different dissociation product that works more efficiently while not causing higher NSC death rate?
- And how you handle the pipette? as to me it's very time-consuming to really dissociate the spheres.
Thank you all in advance!
it is a good idea to ask, as trypsin is not suitable for this task. Use papain, for example see our earlier paper PMID: 19609935, DOI: 10.1002/stem.177 . Trituration ("mechanically pipetting slowly") should not be used, this is an old approach, causing a higg## of cells to die. N.spheres have more and less differentiated cells, more differentiated cells will die (high number). Others use accutase, but papain (with or without very gentle trituration) is #1 choice. You can also have the n.spheres attach and release neural cells (if the goal is to culture) or embed in OCT and generate cryosections and do IHC (if the goal is counting % of different cell types). If you are doing flow - definitely do not do trypsin, as it digests and destroys many key epitopes on the surface of neural cells (see Maric and Barker papers for this). Good luck
Igor O. Nasonkin Hi Igor, thank you very much for your share! I checked with your paper and I have one concern that, as papain is normally used for tissue dissociation, would it be too harsh for iPSC derived NPCs?
I know little about papain, as your paper mentioned 20 U/ml works effectively with E13 embryos, which concentration do you recommend with neuronal spheroids?
Also, do you still mechanically pipette after the treatment? If so, how long does it take to make single cells? is it easier to dissociate after papain treatment than other enzymes?
good questions and comments, too. papain is a standard reagent for dissociating neurospheres, neural tissue and neural rosettes. Consider it a miniature chunk of neural tissue. There's nothing magical about the fact it comes from iPSCs, its just a piece of neural tissue derived in a dish
Igor O. Nasonkin Thank you Igor, you convinced me, ready to purchase now. Would be super nice if you could give some comments to the following two questions too:
Your paper mentioned 20 U/ml papain works effectively with E13 embryos, which concentration do you recommend with neuronal spheroids?
Also, do you still mechanically pipette after the treatment? If so, how long does it take to make single cells? is it easier to dissociate after papain treatment than other enzymes?
Hi,
I understand that you ask about the dissociation of embryoid bodies after neuroectoderm formation?
After induction of neuroectoderm formation (in embryoids bodies), 3D aggregates are not dissociated into single cells. After differentiation of the hiPSC-derived EBs to NSC, the EBs adhere to the culture plate (without dissociation) and then the differentiated cells migrate from them. After 3-6 passage NSC are typically use to obtain next stage of neural differentiation: neural progenitors and then neurons.
Good luck!
use ROCK inhibitor during dissociation and seeding for 1-2 days. Then remove ROCK (neural cells change with continuous exposure to ROCK). Use no trituration, spheres will fall apart into clumps, seed clumps, not single cells. Cells survive much better and do not differentiate as much when they are seeded at high density (clumps provide this feeling of high density). Read about Notch signaling and impact on differentiation for multipotential neural precursors. This is easy stuff. Use 0.1% BSA in media (for human cells it is better to use HSA but for nonclinical work BSA works ok) to make cells less sticky. Check our older papers for recepie (in suppl. data) Article Long-Term, Stable Differentiation Of Human Embryonic Stem Ce...
orArticle Characterization of Three-Dimensional Retinal Tissue Derived...
. Good questions. Use Neurobasal media rather than DMEM/F12 or at least supplement rich media with B27 or N2+B27 and NEAA, dont use serum. We provide good and hepful protocol- Badges
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