CM selection:
- Once CMs are contracting, i.e., from day 7 to day 11, cells are kept in RPMI supplemented with 1XB27(+insulin).
- To increase cardiomyocyte purity, cells are cultured in RPMI without glucose for three days (day 11 to day 14). (5mM sodium lactate supplement is recommended as an alternative energy source for CMs).
- Change the media to RPMI supplemented with 1XB27 (day 14 to day 16)
- On day 16, the iPSC-CMs are dissociated with TrypLE 10x and seeded in 6-well Matrigel-coated plates at a density of 2-3x106 per well in RPMI with 1XB27 containing 10% KOSR and ROCK inhibitor Y-27632 (replating media).
- After 2 days, cells are cultured in RPMI with 1XB27 without glucose for 3 days. Post-starvation, maintain them on RPMI B27+ for 2 days before switching to 3 ml of a metabolism-based maturation medium.
Protocol cartoon
CM maturation media:
Maturation media was composed in DMEM without glucose supplemented with:
- 3mM glucose - (moles/liter) - In the cupboard (genomics)
- 10mM L-lactate
- 5mg/ml Vitamin B12 - In fridge (cell culture room)
- 0.82mM Biotin - In fridge (cell culture room)
- 5mM Creatine monohydrate - In the cupboard (genomics)
- 2mM Taurine - In the cupboard (genomics)
- 2mM L-carnitine - In the cupboard (genomics)
- 0.5mM Ascorbic acid - In the cupboard (genomics)
- 1x NEAA - In fridge (cell culture room)
- 0.5% (w/v) Albumax - In fridge (cell culture room)
- 1x B27 and 1% KOSR
CM freezing from Burridge et al. 2015 (VV edit)
- For cells at day 15
- Frozen in 90% FBS and 10% DMSO, FBS recomended, 2mil cells per mL
- After thawing, spin in a medium with 20%FBS (one vial, 10mL of media) (expect viability 60%)
- Thaw in density 250k/cm2 (1million per 12 well) (they recomend their CDM medium, would try in 10%FBS in B27 (+) with RI 5uM)
- Change media after 48 hours for B27 (+)