I used hiPSC differentiation to obtain large tracts of beating cardiomyocytes, but upon digestion, they all died, or were very weak and clumped.
Hello Xie!
Hope I got your point correctly.
To avoid CMs clumps during splitting/digestion, you have to filter the cardiomyocytes after centrifugating the cells and pipet the suspension up and down many times until you get a single-cell suspension.
After that, the cells should be recovered within 4-5 days.
I used to use this protocol for CMs splitting:
1. Aspirate old medium and gently wash with PBS.
2. Cell detachment: Add 1 ml of TrypLE per well and incubate at 37°C for 10–15mins.
3. Meanwhile, prepare the digestion medium (45 mL RPMI+B27 + 5 mL Knockout serum + 500 ul Revita cells).
4. Make sure of cell dissociation.
5. Add 1 ml of digestion medium per well on the top of TrypLE and collect the cell suspension into a falcon tube.
6. Centrifuge for 5 min at 500g. Meanwhile, remove the coating medium (geltrex) from the new plates and add 1 mL of digestion medium per well.
7. After centrifugation is complete, aspirate the medium without disturbing the pellet.
8. Add the required amount of digestion medium to the cell pellet.
9. Resuspend the cells by pipetting up and down until the suspension is colloidal and homogenous.
10. Filter the suspension.
11. Add 1 mL cell suspension to each well and disribute the suspension equaly.
Hopefully this would be helpful for you!
Aya Abouelalamin
Hello, Professor!
Thank you very much indeed. I will try it according to your method and hope to have some good news. In addition, I would like to ask whether the working concentration of TrypLE you use is 1x and what is the Cat. NO. , looking forward to your reply, thank you again.
Hello!
You are most welcome. I am using TrypLE™ Select Enzym (10x), no phenolred, Cat. No. A1217701.
Best of luck with your iPSC-CMs :))
Aya Abouelalamin
Thank you for your patient response and thank you again!
Dear Xie,
In the initial stage of differentiation of hiPS cells to CM, it is best to continue to passage cells in clumps. Then, when you start passaging with single cells, use Gentle Dissociation Reagent or Accutase and add ROCK inhibitor to the culture medium.
To increase cell viability, ROCK Inhibitor can also be added in the initial stages of passaging, when cells are passaged in colonies.
Good luck!
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