I am culturing human induced pluripotent stem cells for differentiation into cardiomyocytes via the Wnti-directed protocol attached here. After passaging into a 12 well plate to start the differentiation, the iPSCs should be 100% confluent the next day so that differentiation can be started. But it takes mine 2-3 days to reach full confluency, and they rapidly lose confluency as I start the differentiation protocol. By around day 5 of the protocol, they have essentially all died off. Unsure what I'm doing wrong and would welcome any theories or advice. I was using ECM Gel but am now using Matrigel. I use mTesR media with ROCKi added for the first 24h only.
Hi Suh Hee Cook
what is the concentration of Wnt activator and Wnt inhibitor you are applying to the cells? Maybe there is something that is going wrong here.
Secondly, I would highly recommend using basal media which is RPMI+HEPES+GlutaMAX medium for the differentiation. I have noticed GlutaMAX medium works much better than RPMI containing L-Glu.
If both the above points are fine then my next suggestion would be to optimize the seeding density and confluency at which you will start the differentiation for the particular cell line that you are using.
It is not necessary that the iPSC differentiation will work best/better at 100% confluency. In my experience, the best cell density for differentiation can vary amongst different iPSC lines.
Good luck with troubleshooting!
Hi Suh Hee Jun,
My name is Effie and I am a Product Manager at STEMCELL Technologies.
Here are my suggestions for troubleshooting your cardiomyocyte differentiation:
-Seed less cells to reach 100% confluency at day 4 (day 4 worked better for me). As Shambhabi Chatterjee mentioned you would need to optimize seeding density for each of your cell lines.
- Start the differentiation the day that your cells are 90-100% confluent instead of waiting for 5 days as this would cause your cells to spontaneously differentiate and/or multilayer. Ideally your cells would be confluent on day 4/5 after you optimize your seeding density.
- Make sure your cells are evenly distributed when you are seeding them or consider performing your differentiation in 6 well plates, as the cells could be more evenly distributed in a 6 well plate format.
It's normal to see some cell death with Wnt activation, however definitely not more than 10-15%, so I would suggest decreasing the CHIR99021 concentration (if using the small molecules approach). However, I would first optimize the cell seeding density (see comments above) and then CHIR99021 concentration.
Alternatively, you could also try STEMCELL Technologies STEMdiff™ Ventricular Cardiomyocyte Differentiation Kit which is optimized for cardiomyocyte differentiation.
I hope this helps!
Kind Regards,
Effie
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