Tina:Cardiomyocyte differentiation protocols

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Monolayer-directed cardiomyocyte differentiation protocol[edit]

Christian Metallo Lab Protocol[edit]

1. Prepare cells on Matrigel-coated plates. Wait until cells reach 100% confluency.

2. [Day 0]

Prepare RPMI/B-27-A medium.

Component Volume
RPMI 1640 medium, without Gln 49 ml
B-27 supplement minus insulin 1 ml
Total 50 mL

Aspirate culture medium. Add 2 ml RPMI/B-27-A medium per well of a 12-well plate (or 1 ml medium per well of a 24-well plate). Add CHIR99021 stock into RPMI/B-27-A medium (final conc. 12 uM CHIR99021).


3. [Day 1] Each day is an exact 24 hrs period.

Aspirate medium. Add 2 ml RPMI/B-27-A medium per well of a 12-well plate. No CHIR99021 required.

4. [Day 2]

Add DMK stock into culture medium (final conc. 0.5 mM DMK). Do not change the basal RPMI/B-27-A medium.

5. [Day 3]

Prepare Combined Medium.

Component Volume
Spent medium 1 ml
Fresh RPMI/B-27A with 0.5 mM DMK) 1 ml
IWP2 2 ul of 5mM stock; final conc. 5uM
Total 2 mL

Shake the plate back and forth gently, aspirate the remaining medium and cell debris. Add 2ml Combined Medium per well.

6. [Day 4]

Add DMK stock into culture medium (final conc. 0.5 mM DMK). Do not change the basal Combined Medium.

7. [Day 5]

Aspirate medium. Add 2 ml RPMI/B-27-A medium per well. No other supplements required.

8. [Day 6]

Add DMK stock into culture medium (final conc. 0.5 mM DMK). Do not change the basal RPMI/B-27-A medium.

9. [Day 7]

Prepare RPMI/B-27-B medium (Insulin Plus formula).

Component Volume
RPMI 1640 medium, without Gln 49 ml
B-27 serum free supplement 1 ml
Total 50 mL

Aspirate medium. Add 2 ml RPMI/B-27-B medium per well.

10. Change new RPMI/B-27-B medium every 3 days.Robust spontaneous contraction should occur by Day 12-15 (as early as Day 9). Cells can maintain its beating phenotype for months.