I currently run into the issue that our iPSC with a TET-O NGN2 construct seem to die from day 7 after conversion with limited cells alive by day 9. I have observed the issue in 4 individual lines all around the same time. I used the following protocol.
Article Rapid Neuronal Differentiation of Induced Pluripotent Stem C...
we do not add the astrocytes to support the culture and leave therefore the FBS out as this is reported to support the astrocytes.
We use a cell density of 40.000 cm2.
Based on morphology it seems that from day 3 onwards in neurobasal medium the cells seem to experience stress.
composition of neurobasal medium (day 3)
neurobasal medium + 1% p/s + 1% glutamax
BDNF - final conc. 10 ng/ml
NT3 - final conc. 10 ng/ml
puromycin - final conc. 1 ug/ml
B27-plus , final conc. 1x
doxycycline - final conc. 4 ug/ml
we treat cells with Ara-C for 1 day on day 3-4, wash it and replace it with new medium. from day 5 onwards we refresh every other day 50% of the medium.
we tested the following and can rule this out.
- glutamax concentration in neurobasal 2mM - 0,5 mM. all ok. best 1 or 2 mM.
- neurobasal medium compared to DMEM-F12 + supplements. neurobasal morphology nicer. still all died.
- b27 v.s. B27 plus supplement - higher viability with B27 plus supplement
- 10-20 ug/ml laminin coating - no difference.
please, if you have any idea i would really like your input.
much appreciated for thinking along!
Best wishes,
Anouk
Hi Anouk!
I am experiencing a similar issue with when I use this protocol. I also don't add astrocytes or FBS. I have been adding laminin (0.2 ug/mL) to the neurobasal medium throughout the differentiation process because I found it helps keep the cells attached to the matrix.
After doing some more reading, I think the issue might be excitotoxicity. This can cause the neurites to fragment. I suggest trying less frequent media changes to help with this. I also use less doxycycline (2ug/mL) to reduce toxicity due to the addition of this antibiotic.
Hi Sterline P. Arjano,
Sorry to hear you also experience similar issues. Thank you for your input! Adding laminin is definitely something we can add to help the neurons. We have indeed also lowered the doxycycline in the past and we did not observed changes so we remained with the original protocol, where less worked indeed fine.
I think you might be right about the excitotoxicity. We currently work on adding human astrocytes to our cultures to hopefully support the neuronal networks and homeostasis.
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